MiRNA molecule defined by its source and  its diagnostic and  therapeutic uses  in diseases or conditions associated with  EMT

ABSTRACT

The invention relates to the diagnostic and therapeutic uses of a miRNA molecule or an equivalent thereof wherein a source of said miRNA molecule or equivalent thereof comprises at least 80 nucleotides and comprises a motif having at least 98% identity with the motif represented by SEQ ID NO:1 or a source thereof in a disease and condition associated with EMT (Epithelial to Mesenchymal Transition).

FIELD OF THE INVENTION

The invention relates to the diagnostic and therapeutic uses of a miRNAmolecule defined by its source later on in diseases and conditionsassociated with EMT (Epithelial to Mesenchymal Transition).

BACKGROUND OF THE INVENTION

Most solid tumors are epithelial in origin (i.e. carcinomas). A loss ofepithelial cell markers (e.g. E-cadherin) and gain of mesenchymal cellmarkers (e.g. N-cadherin and Vimentin) has been observed in patienttumor samples, including prostate cancer (1). Cancer cells candedifferentiate through this so-called Epithelial to MesenchymalTransition (EMT). During EMT, intercellular cell junctions are brokendown, thereby giving tumor cells the ability to migrate and invade intothe surrounding tissue or through blood vessel walls. Such phenotypicchanges are thought to play a major role in dissemination of the diseaseand ultimately lead to disease progression, which is often associatedwith poor prognosis for the patients (2; 3).

Loss of E-cadherin expression is considerd as a molecular hallmark ofEMT. EMT in tumor cells results from a transcriptional reprogramming ofthe cell. In particular the transcriptional repression of the E-cadherin(CDH1) gene promoter has been shown to trigger the EMT phenotype. TheE-cadherin protein is one of the most important cadherin moleculesmediating cell-cell contacts in epithelial cells/tissues. CDH1 isrepressed by binding of the transcriptional repressors, SNAI1, SNAI2,TCF3, TWIST, ZEB1, ZEB2 or KLF8 (4-7), to three so-called E-boxes in theCDH1 proximal promoter region (8-10). Inhibiting the binding of theserepressors to the CDH1 promoter can revert EMT, also called mesenchymalto epithelial transition (MET), and inhibits tumor cell invasion andtumor progression (11).

Recently, the expression of several microRNAs has been shown to belinked with EMT (12). By comparing microRNA expression profiles of cellswith an epithelial and (induced) mesenchymal phenotype, members of themiR-200 family (miR-141, miR-200a/b/c, and miR-429) and miR-205 wereidentified as EMT-associated miRs (13-15). The target genes of theEMT-associated microRNAs of the miR-200 family were shown to be ZEB1 andZEB2. MicroRNAs targeting the other known transcriptional repressors ofCDH1 (i.e. SNAI1, SNAI2, TCF3 and TWIST1) have not yet been found. Theidentification of these microRNAs in an expression profile of cellswhich have undergone EMT does not necessarily mean that these microRNAsare involved during EMT.

There is currently no effective known medicament that may be used forspecifically preventing, treating, reverting and/or delaying a diseaseor condition associated with EMT in a subject. The only standardtreatments comprise chemotherapy, radiotherapy, surgery. Particularly,the identification of patients that will or have already developedmetastases and/or early treatment of patients with tumors expressing EMTmarkers, such as expression of mesenchymal Cadherin and/or lowerexpression of E-Cadherin could contribute to better disease free andoverall survival. Therefore, there is still a need for diagnosticmarkers for EMT and for new treatments of disease or conditionsassociated with EMT.

DESCRIPTION OF THE INVENTION

In a first aspect, there is provided a miRNA molecule or an equivalentthereof wherein a source of said miRNA molecule or equivalent thereofcomprises at least 80 nucleotides and comprises a motif having at least98% identity with the motif represented by SEQ ID NO:1 or a compositioncomprising said miRNA molecule or equivalent thereof or said sourcethereof for use as a medicament for preventing, treating, reverting,curing and/or delaying a disease or a condition associated with EMT.

MicroRNAs (miRNAs) are small RNAs of 17-25 nucleotides, which functionas regulators of gene expression in eukaryotes. miRNAs are initiallyexpressed in the nucleus as part of long primary transcripts calledprimary miRNAs (pri-miRNAs). Inside the nucleus, pri-miRNAs arepartially digested by the enzyme Drosha, to form 65-120 nucleotide-longhairpin precursor miRNAs (pre-miRNAs) that are exported to the cytoplasmfor further processing by Dicer into shorter, mature miRNAs, which arethe active molecules. In animals, these short RNAs comprise a 5′proximal “seed” region (nucleotides 2 to 8) which appears to be theprimary determinant of the pairing specificity of the miRNA to the 3′untranslated region (3′-UTR) of a target mRNA. A more detailedexplanation is given in the part dedicated to general definitions.

Each of the definitions given below concerning a miRNA molecule, a miRNAequivalent or a miRNA source is to be used for each of the identifiedmiRNAs or miRNA equivalent or miRNA sources of this application:miRNA-124-1, miRNA-206, miRNA181a-1, miRNA-141, miRNA-200a, miRNA-200b,miRNA-200c, miRNA-429, miRNA-205, miRNA518b, miRNA520f, miRNA524 andsources thereof, further including a source comprising at least 80nucleotides and comprising a motif having at least 98% identity with themotif represented by SEQ ID NO:1. Preferred mature (as identified inTable 3), seed (as identified in Table 5) isomiRs (as identified inTable 6) or source sequences (as identified in Tables 2 (RNA precursor)or 4 (DNA encoding a RNA precursor)) of said miRNA molecule orequivalent thereof respectively are identified in corresponding tables.

Within the whole text of the application unless otherwise indicated, amiRNA may also be named a miRNA molecule, a miR, or an equivalentthereof or a source or a precursor thereof. A preferred equivalent is amature, an isomiR, or a mimic. Each sequence identified herein may beidentified as being SEQ ID NO as used in the text of the application oras corresponding SEQ ID NO in the sequence listing.

In the context of the invention a miRNA molecule or an equivalent or amimic or an isomiR thereof may be a synthetic or natural or recombinantor mature or part of a mature miRNA or a human miRNA or derived from ahuman miRNA as further defined in the part dedicated to the generaldefinitions. A human miRNA molecule is a miRNA molecule which is foundin a human cell, tissue, organ or body fluids (i.e. endogenous humanmiRNA molecule). A human miRNA molecule may also be a human miRNAmolecule derived from an endogenous human miRNA molecule bysubstitution, deletion and/or addition of a nucleotide. A miRNA moleculeor an equivalent or a mimic thereof may be a single stranded or doublestranded RNA molecule.

Within the context of the invention, a preferred miRNA molecule or anequivalent or a mimic or an isomiR thereof is such that a source of saidmiRNA molecule or equivalent or mimic or isomiR thereof comprises orconsists of at least 80 nucleotides and comprises a motif having atleast 98% identity with the motif represented by SEQ ID NO:1 is furtherpreferably defined as follows.

SEQ ID NO: 1 is as follows:

UCAnGCUGUGnCCCUnnAnAGGGAAGCnCUUUCUnUnGUCnnAAnGAAAAnnAnGnGCUnCCnUUUnGAGnnUUACnGUUUG

In the motif represented by SEQ ID NO:1, n may be any base A, U, C or G.

In a further preferred embodiment, there is provided a miRNA molecule oran equivalent or a mimic or a isomiR thereof such that a source of saidmiRNA molecule or equivalent or mimic or isomiR thereof comprises atleast 80 nucleotides and comprises a motif having at least 98% identitywith the motif represented by SEQ ID NO:1. More preferably, the identityis at least 99% or 100%. In a preferred embodiment, said source is aprecursor of a miRNA-518b, a miRNA-520f or a miRNA-524 molecule or anequivalent or a mimic or a isomiR thereof. Preferred sources andprecursors are later defined herein.

The invention therefore relates to a miRNA molecule or an equivalent ora mimic or an isomiR thereof or a source thereof or a compositioncomprising said miRNA molecule or equivalent or mimic or isomiR thereofor source thereof, as defined in coming paragraphs, wherein the miRNAmolecule is a miRNA-518b, miRNA-520f and/or miRNA-524 or an equivalentor a mimic or an isomiR thereof or a source thereof. Preferred miRNAmolecule, equivalent, mimic and isomiR are later defined herein.

In a preferred embodiment, an equivalent of a miRNA-518b, a miRNA-520for of a miRNA-524 is a human miRNA molecule. A human miRNA molecule is amiRNA molecule which is found in a human cell, tissue or organ. A humanmiRNA molecule may also be a human miRNA molecule derived from anendogenous human miRNA molecule by substitution, deletion and/oraddition of a nucleotide. In this context, a “nucleotide” may mean 1, 2,3, 4, 5 or more nucleotides. A preferred equivalent of a miRNA-518b, amiRNA-520f or of a miRNA-524 molecule is not an mml-mir-519a or anmml-mir-520c miRNA molecule as identified hereafter. A preferred sourceor precursor of a miRNA-518b, a miRNA-520f or of a miRNA-524 molecule isnot a source or a precursor of an mml-mir-519a or an mml-mir-520c miRNAmolecule as identified hereafter. Preferred disclaimed mature andprecursor sequences of mml-mir-519a are identified as SEQ ID NO: 108 and109. Preferred mature and precursor sequences of mml-mir-520c areidentified as SEQ ID NO: 110 and 111.

In a preferred embodiment, a miRNA molecule or equivalent or mimic orisomiR thereof comprises at least 6 of the 7 nucleotides present in theseed sequence of said miRNA molecule or equivalent or mimic or isomiRthereof (Table 5 shows preferred seed sequence of each of the miRNAsmolecule identified herein). Preferably in this embodiment, a miRNAmolecule or an equivalent or a mimic or isomiR thereof can be from 6, 7,8, 9, 10, 11, 12 to 30 nucleotides in length and more preferablycomprises at least 6 of the 7 nucleotides present in the seed sequenceof said miRNA molecule or equivalent thereof. Even more preferably amiRNA molecule or an equivalent or a mimic or isomiR thereof is from 15to 28 nucleotides in length and more preferably comprises at least 6 ofthe 7 nucleotides present in the seed sequence. Even more preferably amiRNA molecule has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30nucleotides or more and preferably comprises at least 6 of the 7nucleotides present in the seed sequence.

In each of these embodiments, a mirRNA molecule or an equivalent or amimic or isomiR thereof may comprise the 7 nucleotides of the seedsequence as identified in table 5. Even more preferably a miRNA moleculehas a length of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more andcomprises the 7 nucleotides present in the seed sequence as identifiedin table 5.

Accordingly, a preferred miRNA-520f molecule or equivalent or mimic orisomiR thereof comprises at least 6 of the 7 nucleotides present in theseed sequence identified as SEQ ID NO: 104 or 105 and more preferablyhas a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.

Accordingly, a preferred miRNA-518b molecule or equivalent or mimic orisomiR thereof comprises at least 6 of the 7 nucleotides present in theseed sequence identified as SEQ ID NO: 103 and more preferably has alength of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.

Accordingly, a preferred miRNA-524 molecule or equivalent or mimic orisomiR thereof comprises at least 6 of the 7 nucleotides present in theseed sequence identified as SEQ ID NO: 106 or 107 and more preferablyhas a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.

In another preferred embodiment, a miRNA molecule or an equivalent ormimic or isomiR thereof comprises at least 6 of the 7 nucleotidespresent in a given seed sequence as identified in table 5 as SEQ ID NO:87-107 and has at least 80% identity over the whole mature sequence(Table 3 shows preferred mature sequences of each of the miRNAsidentified herein). Preferably, identity is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or highersuch as 96%, 97%, 98%, 99% or 100%.

Alternatively, preferably in this embodiment, a miRNA molecule or anequivalent or a mimic or an isomiR thereof has a length of not more than6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40nucleotides, comprises at least 6 of the 7 nucleotides present in agiven seed sequence as identified in table 5 as SEQ ID NO: 87-107 andhas at least 80% identity over the whole mature sequence as identifiedin table 3 as SEQ ID NO: 2-21.

Preferably, identity is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another preferred embodiment, an isomiR of a miRNA molecule has atleast 80% identity over the whole isomiR sequence (Table 6 showspreferred isomiR of each of the mature miRNAs identified as SEQ ID NO:118-162). Preferably, identity is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or higher such as 96%,97%, 98%, 99% or 100%. Preferably in this embodiment, an isomiR of amiRNA molecule or an equivalent or a mimic thereof has a length of atleast 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.

Identity may be assessed using several ways as defined later herein.However, in a preferred embodiment, identity means identity percentageand is calculated by the number of equal nucleotides between subject andquery, divided by the total length of the query, and multiplied by 100.

Accordingly, a preferred miRNA-520f molecule or equivalent or mimic orisomiR thereof comprises at least 6 of the 7 nucleotides present in theseed sequence identified as SEQ ID NO: 104 or 105 and/or has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,945, 95%, 96%, 97%, 98%, 99% or 100% identity over SEQ ID NO: 19, 118,119 and/or 120 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more.

Accordingly, a preferred miRNA-518b molecule or equivalent or mimic orisomiR thereof comprises at least 6 of the 7 nucleotides present in theseed sequence identified as SEQ ID NO: 103 and/or has at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 945, 95%,96%, 97%, 98%, 99% or 100% identity over SEQ ID NO: 18, 121, 122 and/or123 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 nucleotides or more.

Accordingly, a preferred miRNA-524 molecule or equivalent or mimic orisomiR thereof comprises at least 6 of the 7 nucleotides present in theseed sequence identified as SEQ ID NO: 106 or 107 and/or has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,945, 95%, 96%, 97%, 98%, 99% or 100% identity over SEQ ID NO: 20, 21,124, 125, 126, 127 and/or 128 and/or has a length of at least 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides ormore.

Another preferred miRNA molecule or equivalent or mimic or an isomiRthereof has at least 80% identity with a seed sequence (as identified inTable 5 as SEQ ID NO: 87-107) or with a mature sequence (as identifiedin Table 3 as SEQ ID NO: 2-21) or with a precursor sequence (asidentified in Table 2 as SEQ ID NO: 22-35) or with a DNA encoding an RNAprecursor (as identified in Table 4 as SEQ ID NO: 36-47) or with anisomiR sequence (as identified in Table 6 as SEQ ID NO: 118-162).Identity may be at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.Identity is preferably assessed on the whole SEQ ID NO as identified ina given Table. However, identity may also be assessed on part of a givenSEQ ID NO. Part may mean at least 50% of the length of the SEQ ID NO, atleast 60%, at least 70%, at least 80%, at least 90% or 100%.

An equivalent may be an isomiR or a mimic. A precursor sequence mayresult in more than one isomiR sequences depending on the maturationprocess (see for example miRNA-520f or miRNA-518b or miRNA-524 where incertain tissues, multiple isomiRs have been identified (Table 6: SEQ IDNO:118-128). A mimic is a molecule which has a similar or identicalactivity with a miRNA molecule. In this context a similar activity isgiven the same meaning as an acceptable level of an activity.

Each of the miRNA molecules or equivalents or mimics or isomiRs thereofas identified herein has an acceptable level of an activity of a givenmiRNA they derive from. An acceptable level of an activity is preferablythat said miRNA or equivalent or mimics or isomiRs thereof is still ableto exhibit an acceptable level of said activity of said miRNA. Anactivity of a given miRNA or an equivalent thereof is for example theability to induce a detectable MET as later defined herein. Anacceptable level of an activity is preferably at least 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100% of the activity of the miRNA they derivefrom. Such activity may be as measured in a bladder cell of anindividual or in vitro in a cell by comparison to the activity of themiRNA they derive from. The assessment of the activity may be carriedout at the mRNA level, preferably using RT-qPCR. The assessment of theactivity may be carried out at the protein level, preferably usingWestern blot analysis or, immunohistochemistry or immunofluorescenceanalysis of cross-sections.

The assessment of the activity may be carried out using cells expressinga CDH1 promotor driven firefly luciferase construct and measuringluciferase activity.

A preferred activity of any of the miRNA molecule or equivalent thereofas identified herein (i.e. miRNA-124-1, miRNA-206, miRNA181a-1,miRNA-141, miRNA-200a, miRNA-200b, miRNA-200c, miRNA-429, miRNA-205,miRNA-518b, miRNA-520f, miRNA-524) or a preferred activity of a miRNAmolecule or equivalent thereof identified by a preferred source (i.e. amiRNA molecule or an equivalent thereof wherein a source of said miRNAmolecule or equivalent thereof comprises at least 80 nucleotides andcomprises a motif having at least 98% identity with the motifrepresented by SEQ ID NO:1) is to induce a detectable MET in a subjectas later defined herein.

A source of a miRNA molecule or a source of an equivalent of a miRNAmolecule, mimic, isomiR may be any molecule which is able to induce theproduction of a miRNA molecule or of an equivalent thereof such as amimic or isomiR as identified herein and which comprises a hairpin-likestructure and/or a double stranded nucleic acid molecule. The presenceof a hairpin-like structure, may be assessed using the RNAshapes program(Steffen P., et al, (2006), Bioinformatics, 22: 500-503) using slidingwindows of 80, 100 and 120 nt or more. The presence of a hairpin-likestructure is usually present in a natural or endogenous source of amiRNA molecule whereas a double-stranded nucleic acid molecule isusually present in a recombinant or synthetic source of a miRNA moleculeor of an equivalent thereof.

A source of a miRNA molecule or of an equivalent or a mimic or an isomiRthereof may be a single stranded, a double stranded RNA or a partiallydouble stranded RNA or comprise three strands, an example of which isdescribed in WO2008/10558. As used herein partially double strandedrefers to double stranded structures that also comprise single strandedstructures at the 5′ and/or at the 3′ end. It may occur when each strandof a miRNA molecule does not have the same length. In general, suchpartial double stranded miRNA molecule may have less than 75% doublestranded structure and more than 25% single stranded structure, or lessthan 50% double stranded structure and more than 50% single strandedstructure, or more preferably less than 25%, 20% or 15% double strandedstructure and more than 75%, 80%, 85% single stranded structure.Alternatively, a source of a miRNA molecule or of an equivalent or amimic or an isomiR thereof is a DNA molecule encoding a precursor of amiRNA molecule or of an equivalent or a mimic or an isomiR thereof.Preferred DNA molecules in this context are identified in table 4 as SEQID NO: 36-47. The invention encompasses the use of a DNA moleculeencoding a precursor of a miRNA molecule that has at least 80% identitywith said sequence as identified in table 4. Preferably, the identity isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Preferably in thisembodiment, a DNA molecule has a length of at least 50, 55, 60, 70, 75,80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides ormore and has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with aDNA sequence as identified in table 4 as SEQ ID NO: 36-47.

Accordingly, a preferred source of a miRNA-520f molecule has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 45 and/orhas a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130,150, 200, 250, 300, 350, 400 nucleotides or more.

Accordingly, a preferred source of a miRNA-518b molecule has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 44 and/orhas a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130,150, 200, 250, 300, 350, 400 nucleotides or more.

Accordingly, a preferred source of a miRNA-524 molecule has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 46 and/orhas a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130,150, 200, 250, 300, 350, 400 nucleotides or more.

The induction of the production of a given miRNA molecule or of anequivalent thereof or of a mimic or an isomiR thereof is preferablyobtained when said source is introduced into a cell using one assay asdefined below. Cells encompassed by the present invention are later ondefined.

A preferred source of a miRNA molecule or of an equivalent thereof or ofa mimic or an isomiR thereof is a precursor thereof, more preferably anucleic acid encoding said miRNA molecule or an equivalent thereof or ofa mimic or an isomiR thereof. A preferred precursor is anaturally-occurring precursor. A precursor may be a synthetic orrecombinant precursor.

A preferred precursor of a given miRNA molecule is identified in table 2as SEQ ID NO: 22-35. The invention encompasses the use of a precursor ofa miRNA molecule or of an equivalent thereof that has at least 80%identity with said sequence. Preferably, identity is at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%. Preferably in this embodiment, an RNAmolecule has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95,100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and has atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence asidentified in table 2 as SEQ ID NO: 22-35.

Accordingly, a preferred source of a miRNA-520f molecule has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 33 and/orhas a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130,150, 200, 250, 300, 350, 400 nucleotides or more.

Accordingly, a preferred source of a miRNA-518b molecule has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 32 and/orhas a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130,150, 200, 250, 300, 350, 400 nucleotides or more.

Accordingly, a preferred source of a miRNA-524 molecule has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 34 and/orhas a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130,150, 200, 250, 300, 350, 400 nucleotides or more.

Preferred sources or precursors have been defined later herein. Apreferred source includes or comprises an expression constructcomprising a nucleic acid, i.e. DNA encoding said precursor of saidmiRNA, more preferably said expression construct is a viral gene therapyvector selected from gene therapy vectors based on an adenovirus, anadeno-associated virus (AAV), a herpes virus, a pox virus and aretrovirus. A preferred viral gene therapy vector is an AAV orLentiviral vector. Other preferred vectors are oncolytic viral vectors.Such vectors are further described herein below. Alternatively, a sourcemay be a synthetic miRNA molecule or a chemical mimic as further definedin the part dedicated to general definitions.

The detection of the presence of a miRNA molecule or of an equivalentthereof such as a mimic or an isomiR may be carried out using anytechnique known to the skilled person. The assessment of the expressionlevel or of the presence of a miRNA molecule or of an equivalent thereofis preferably performed using classical molecular biology techniquessuch as (real time) qPCR, microarrays, bead arrays, RNAse protectionanalysis or Northern analysis or cloning and sequencing. The skilledperson will understand that alternatively or in combination with thequantification of a miRNA molecule or of an equivalent thereof, thequantification of a substrate of a corresponding miRNA molecule or of anequivalent thereof or any compound known to be associated with afunction of said miRNA molecule or of said equivalent thereof or thequantification of a function or activity of said miRNA molecule or ofsaid equivalent thereof using a specific assay is encompassed within thescope of the invention.

Preferred compositions and formulations are all defined later herein. AmiRNA molecule or an equivalent thereof or a mimic or an isomiR thereofmay be used as such as a naked molecule, with or without chemicalmodifications, or encapsulated into a particle or conjugated to amoiety. A preferred composition comprises a miRNA molecule or anequivalent thereof or a mimic or an isomiR thereof encapsulated into ananoparticle or a liposomal structure. A miRNA molecule or equivalentthereof or a mimic or an isomiR thereof may be an aptamer-miRNA hybrid.A miRNA molecule or equivalent thereof may be an aptamer-miRNA hybrid.An aptamer-miRNA is defined as a miRNA linked to an RNA (or DNA)oligonucleotide, the latter adopting a conformation that targets theaptamer-miRNA hybrid molecule to a cell-surface protein (e.g. theProstate-Specific Membrane Antigen (PSMA)). The aptamer-tagged miRNA canbe linked to e.g. polyethylene glycol, which increases the chimera'scirculating half-life (Dassie, J. P., et al. Nat. Biotechnol. 27:839-849 (2009)).

Any disease or condition wherein EMT is involved or associated may beprevented, delayed, cured and/or treated with a molecule as definedherein.

Within the context of the invention, Epithelial Mesenchymal Transition(EMT) is an orchestrated series of events in which cell-cell andcell-extracellular matrix (ECM) interactions are altered to allow therelease of epithelial cells from the surrounding tissue. The epithelialcell cytoskeleton is reorganised to confer the ability of the cell tomove through a three-dimensional ECM via molecular reprogramming of thecell. Molecular reprogramming of an epithelial cell is necessary toachieve a mesenchymal phenotype and involves the downregulation ordecrease of the expression of epithelial proteins, such as E-cadherinand junction proteins such as desmoplakin, claudin and occludin. Inaddition, the expression of mesenchymal proteins is upregulated orincreased, including for example, the expression of ECM proteins such asMMPs and fibronectin and cell surface proteins such as N-cadherin andintegrin αvβó. Transcription factors may also be upregulated orincreased in cells exhibiting a mesenchymal phenotype such as forexample, SNAI1 (also known as SNAIL), TWIST, ZEB1 (also known as δEF1)and ZEB2 (also known as SIP1). Reference to inducing the “transition” ofan epithelial cell to a cell exhibiting a mesenchymal phenotype shouldbe understood as a reference to inducing the genetic, morphologic and/orfunctional changes which are required to change an epithelial cell to acell exhibiting a mesenchymal phenotype of the type defined herein.Reference to inducing mesenchymal to epithelial transition should beunderstood to have the converse meaning.

In a disease or condition of the invention, EMT may be detectable beforethe onset of the disease or condition i.e. before the appearance of asymptom of said disease or condition. It is further encompassed by thepresent invention that EMT is detectable during the development of saiddisease or condition, i.e. after the apparition of a symptom of saiddisease or condition. It is also further encompassed that EMT isdetectable before the onset of the disease or condition and during thedevelopment of said disease or condition.

EMT may be detected using any technique known to the skilled person.Preferably, EMT is assessed by detecting a decrease of the expression ofepithelial E-cadherin and/or an increase of the expression ofmesenchymal vimentin and/or mesenchymal cadherin usingimmunohistochemistry using specific antibodies raised against E-cadherinand/or mesenchymal vimentin and/or mesenchymal cadherin respectively (2,3). N-cadherin (CDH2) and OB-cadherin (CDH11) are two examples ofmesenchymal cadherins. The assessment of the expression is preferablycarried out in a tumor biopsy or section at several time points for agiven subject or at one or several time points for a given subject and ahealthy control. The assessment may be carried out each week, eachmonth. The increase/decrease may therefore be assessed each week, month.Preferably, a decrease of the expression of epithelial E-cadherin meansa significant decrease, preferably a decrease of at least 5% of theexpression using immunohistochemistry. More preferably, a decrease meansa decrease of at least 10%, even more preferably at least 20%, at least30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. Inthis case, no expression is detectable. A 25 fold increase of E-cadherinwas obtained using a miRNA-520f molecule as identified herein (arepresentative example is shown in example 2, FIG. 4B). The effect ofthis miRNA molecule on this marker is quantitatively (at least 1.5 fold)more pronounced than the corresponding effect of miRNA molecule of the200 family that were already known to have an effect on E-cadherin asshown in in example 2, FIG. 4B. We therefore may anticipate that amiRNA-520f molecule as identified herein may be considered as anattractive molecule for a use as identified herein.

Preferably, an increase of the expression mesenchymal vimentin and/ormesenchymal cadherin means a significant increase, preferably anincrease of at least 5% of the expression using immunohistochemistry.More preferably, an increase means an increase of at least 10%, evenmore preferably at least 20%, at least 30%, at least 40%, at least 50%,at least 70%, at least 90%, at least 100%, or at least 150% or more.

A disease or condition wherein EMT is involved or associated ispreferably a disease or condition wherein a dedifferentiation processoccurs. In this dedifferentiation process, a decrease of the expressionof epithelial E-cadherin and/or an increase of the expression ofmesenchymal vimentin and/or mesenchymal cadherin preferably occurs andmay be assessed as explained herein. As an example, EMT may be exhibitedby cancer cells which undergo this process and thereby become metastaticdue to their ability to separate from neighbouring cells and penetrateinto and through surrounding tissues. Therefore, a preferred disease inthis context is a cancer (e.g., malignant, metastatic) or a fibrosis.Cancers of a preferred embodiment of the invention include a cancer ofepithelial origin or a carcinoma or a solid tumor. Cancer cells may befrom the bladder, brain, breast, colon, esophagus, gastrointestine,head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin,stomach, testis, tongue, or uterus. In addition, the cancer mayspecifically be of the following histological type, though it is notlimited to these: neoplasm, malignant; carcinoma; carcinoma,undifferentiated; giant and spindle cell carcinoma; small cellcarcinoma; papillary carcinoma; squamous cell carcinoma; basal cellcarcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillarytransitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellularcarcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoidcystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma;basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma;follicular adenocarcinoma; papillary and follicular adenocarcinoma;nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;endometroid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinouscystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;inflammatory carcinoma; paget's disease of the breast; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; ovarian stromal tumor, malignant; androblastoma, malignant;Sertoli cell carcinoma. In a preferred embodiment, the disease orcondition associated with EMT is a cancer, preferably a bladder orprostate cancer. EMT does not occur in all cancers. In soft tissuesarcomas and leukemias the dedifferentiation process of EMT does notoccur. Therefore in a preferred embodiment, a cancer as identifiedherein is a carcinoma and/or is not a leukemia and/or is not a tissuesarcoma.

EMT may also occur during chronic inflammation or conditions thatpromote sustained tissue disruption which can stimulate fibrosis,thereby compromising tissue integrity and organ function. A fibrosis isalso known as organ fibrosis or organ degeneration (reviewed in ThierryJ. P. et al, 2009, Cell, 139: 871-890). In fibrotic tissues,myofibroblasts accumulate and secrete an excessive amount of collagenthat is deposited as fibers, thereby compromising organ function andleading to its failure. Fibrosis originates from the conversion of asignificant portion of epithelial cells into myofibroblasts through anEMT process (Iwano et al., 2002 J. Clin. Invest. 110:341-50). Initiallydemonstrated in differentiated cells of renal tubules and ducts, it isnow clear that lens epithelium, endothelium, hepatocytes, andcardiomyocytes can all undergo EMT and contribute significantly totissue fibrosis. Each fibrosis wherein EMT is supposed or suspected tooccur is encompassed within the scope of the invention.

A condition or disease associated with EMT may also be poor woundhealing, diabetic renal nephropathy, allograft dysfunction, cataracts ordefects in cardiac valve formation.

There is currently no effective known medicament that may be used forspecifically preventing, treating, reverting, curing and/or delaying adisease or condition associated with EMT in a subject. The inventionencompasses to use a miRNA molecule or an equivalent thereof wherein asource of said miRNA molecule or equivalent thereof comprises at least80 nucleotides and comprises a motif having at least 98% identity withthe motif represented by SEQ ID NO:1 or a composition comprising saidmiRNA molecule or equivalent thereof or a source thereof to this end.Preferred miRNA molecules or equivalents or mimic or isomiR or sourcesthereof have already been defined herein.

This use includes pharmacologically increasing an activity or thesteady-state level of said miRNA molecule or equivalent thereof or ofsaid source thereof in a subject, in a cell of said subject, in a tissueof said subject or in body fluid of said subject.

In this use, an activity or steady-state level of said miRNA, orequivalent thereof or source thereof is increased in order to induce adetectable MET in a subject. A MET, Mesenchymal to Epithelial Transitionis the reverse of an EMT. Therefore, induction of MET is identical toreversion of EMT. Preferably, a MET is assessed by detecting an increaseof the expression of epithelial E-cadherin and/or a decrease of theexpression of mesenchymal vimentin using immunohistochemistry using aspecific antibody raised against E-cadherin, respectively mesenchymalvimentin and/or mesenchymal cadherin (2, 3). The assessment of theexpression is preferably carried out in a tumor biopsy or section atseveral time points for a given subject or at one or several time pointsfor a given subject and a healthy control. The assessment may be carriedout at regular time intervals, e.g. each week, each month. Theincrease/decrease may therefore be assessed regularly, e.g. each week,each month. A MET has been preferably detected when for at least onetime point, an increase of the expression of epithelial E-cadherinand/or an decrease of the expression of mesenchymal vimentin and/ormesenchymal Cadherin has been detected. Preferably, a MET has beendetected when for at least two, three, four, five time points suchincrease of the expression of epithelial E-cadherin and/or decrease ofthe expression of mesenchymal vimentin and/or mesenchymal Cadherin hasbeen detected. Preferably, an increase of the expression of epithelialE-cadherin means a significant increase, preferably an increase of atleast 5% of the expression using immunohistochemistry. More preferably,an increase means an increase of at least 10%, even more preferably atleast 20%, at least 30%, at least 40%, at least 50%, at least 70%, atleast 90%, at least 150% or more.

Preferably, a decrease of the expression mesenchymal vimentin and/ormesenchymal cadherin means a significant decrease, preferably a decreaseof at least 5% of the expression using immunohistochemistry. Morepreferably, a decrease means a decrease of at least 10%, even morepreferably at least 20%, at least 30%, at least 40%, at least 50%, atleast 70%, at least 90%, or 100%. In this case, no expression isdetectable.

An activity or steady-state level of said miRNA molecule or anequivalent or a mimic or an isomiR thereof wherein a source of saidmiRNA molecule or equivalent or mimic or isomiR thereof comprises atleast 80 nucleotides and comprises a motif having at least 98% identitywith the motif represented by SEQ ID NO:1 may be increased at the levelof the miRNA molecule (or equivalent or mimic or isomiR thereof) itself,e.g. by providing said miRNA molecule or equivalent or mimic or isomiRthereof to a subject, preferably to a cell of a subject, or to a tissueof said subject, or to an organ of said subject or to said subject saidmiRNA molecule or equivalent or mimic or isomiR thereof being from anexogenous source. For provision of a miRNA molecule or equivalent ormimic or isomiR thereof from an exogenous source, said miRNA molecule orequivalent or mimic or isomiR thereof may conveniently be produced byexpression of a nucleic acid encoding said miRNA molecule or equivalentor mimic or isomiR thereof or encoding a source of said miRNA moleculeor equivalent or mimic or isomiR thereof in a suitable host cell asdescribed below or as completely synthetic molecules by chemicalsynthesis.

Preferably, however, an activity or steady-state level of a miRNAmolecule or equivalent or a mimic or an isomiR thereof is increased byregulating the expression level of a nucleotide sequence encoding saidmiRNA molecule or equivalent or mimic or isomiR thereof or encoding asource of said miRNA molecule or equivalent or mimic or isomiR thereof.Preferably, the expression level of a nucleotide sequence is regulatedin a cell of said subject or in a tissue of said subject or in thesubject. The expression level of a miRNA molecule or equivalent or mimicor isomiR thereof or a source of said miRNA molecule or equivalent ormimic or isomiR thereof may be increased by introduction of a miRNAmolecule, or equivalent or mimic or isomer thereof, or a source thereof,or an expression construct (or vector) into a cell, tissue, organ orbody fluid of said subject, or in the subject whereby an expressionvector comprises a nucleotide sequence comprising a miRNA molecule orequivalent or a mimic or an isomiR thereof or comprising a source ofsaid miRNA molecule or equivalent or a mimic or an isomiR thereof, andwhereby a nucleotide sequence is under control of a promoter capable ofdriving expression of a nucleotide sequence in said cell, tissue, organ,subject. The expression level of a miRNA molecule or equivalent or amimic or an isomiR thereof or source thereof may also be increased byintroduction of an expression construct into a cell, tissue, organ,subject, whereby a construct comprises a nucleotide sequence encoding afactor capable of trans-activation of an endogenous nucleotide sequenceencoding a miRNA molecule or equivalent or mimic or isomiR thereof.

A use of the invention preferably comprises the step of administering toa subject a therapeutically effective amount of a pharmaceuticalcomposition comprising a nucleic acid construct for increasing theactivity or steady state level of a miRNA molecule or an equivalent or amimic or an isomiR thereof wherein a source of said miRNA molecule orequivalent or mimic or isomiR thereof comprises at least 80 nucleotidesand comprises a motif having at least 98% identity with the motifrepresented by SEQ ID NO:1 or a source thereof as identified herein. Anucleic acid construct may be an expression construct as furtherspecified herein. Preferably, an expression construct is a viral genetherapy vector selected from gene therapy vectors based on anadenovirus, an adeno-associated virus (AAV), a herpes virus, a poxvirus, an oncolytic virus vector and a retrovirus. A preferred viralgene therapy vector is an AAV or Lentiviral vector. Alternatively, a useof the invention preferably comprises the step of administering to asubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a miRNA molecule or an equivalent or a mimic oran isomiR thereof wherein a source of said miRNA molecule or equivalentor mimic or isomiR thereof comprises at least 80 nucleotides andcomprises a motif having at least 98% identity with the motifrepresented by SEQ ID NO:1 or a source thereof as defined herein.

In a use of the invention, a cell, a tissue, an organ or body fluid ispreferably from a subject suspected to have a high risk of having an EMTor of having a disease or condition associated with EMT due for exampleto its age or its genetic background or to its diet. Alternatively, inanother preferred embodiment, use of the invention is applied on a cell,tissue, organ or body fluid from a subject diagnosed as either having apredictive risk for developing later a disease or condition associatedwith EMT. A diagnostic method used is preferably one of the inventionsas described herein. Alternatively, a cell, a tissue or organ to betreated may be selected based on risk of progression of the disease orcondition associated with EMT. Such risk of progression may be assessedusing classical clinic-pathological criteria or biomarker-basedprognosis known to the skilled person. It is also encompassed by theinvention to administer a miRNA molecule or an equivalent or a mimic oran isomiR thereof wherein a source of said miRNA molecule or equivalentor mimic or isomiR thereof comprises at least 80 nucleotides andcomprises a motif having at least 98% identity with the motifrepresented by SEQ ID NO:1 or a precursor thereof or a compositioncomprising said miRNAmolecule or equivalent or mimic or isomiR thereofor source thereof into a tissue or organ of said subject. PreferredmiRNA molecules or equivalents or mimic or isomiR or sources thereofhave already been defined herein. In the invention, a preferred cell,tissue or organ is a cell, tissue or organ that is or comprises abladder or prostate cell or tissue or is or comprises the bladder orprostate as organ.

A treatment of a disease or condition associated with EMT may include atreatment that prevents EMT in a tumor cell that has not yetmetastasized or revert EMT (defined as Mesenchymal to EpithelialTransition) in a tumor cell that has already formed metastases and/or ismigrating from the primary tumor to distant sites in the body.

In another use, the invention mentioned herein may be combined withstandard treatments of disease or condition associated with EMT such aschemotherapy, radiotherapy or surgery. Examples of chemotherapeuticagents are exemplified later herein.

Although gene therapy is a possibility for preventing, treating,reverting and/or delaying a condition or a disease associated with EMT,other possible treatments may also be envisaged. For example, treatmentby “small molecule” drugs to steer certain molecular pathways in thedesired direction, is also preferred. These small molecules arepreferably identified by the screening method of the invention asdefined later herein.

In the context of the invention, preventing, treating, reverting, curingand/or delaying a disease or condition associated with EMT may meanthat:

-   -   At least a symptom of this disease or condition has been        improved, and/or    -   At least a parameter associated with this disease or condition        has been improved.

A symptom may be the presence of metastases as explained below. Aparameter may be the assessment of MET as explained earlier herein. Inthe context of the invention, preventing, treating, reverting, curingand/or delaying a disease or condition associated with EMT may bereplaced by achieving an anti-tumor effect. Unless otherwise indicated,an anti-tumor effect is preferably assessed or detected after at leastone week, two weeks, three weeks, fours weeks, one month, two months,three months, four months, five months, six months or more in a treatedsubject. An anti-tumor effect is preferably identified in a subject as:

-   -   an inhibition of proliferation of tumor cells and/or    -   an increase in the capacity of differentiation of tumor cells        and/or    -   an induction or increased induction of tumor cells death and/or    -   a delay in occurrence of metastases and/or of tumor cell        migration and/or    -   an inhibition or prevention or delay of the increase of a tumor        weight or growth and/or    -   a prolongation of patient survival of at least one month,        several months or more (compared to those not treated or treated        with a control or compared with the subject at the onset of the        treatment).

In the context of the invention, a patient may survive and/or may beconsidered as being disease free. Alternatively, the disease orcondition may have been stopped or delayed. An inhibition of theproliferation of tumor cells may be at least 20%, 30%, 40%, 50%, 55%,60%, 65%, 70% or 75%, or more. Proliferation of cells may be assessedusing known techniques.

An induction of tumor cell death may be at least 1%, 5%, 10%, 15%, 20%,25%, or more. Tumor growth may be inhibited at least 5%, 10%, 20%, 30%,40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Tumor cell death may beassessed using techniques known to the skilled person. Tumor cell deathmay be assessed using MRI (Magnetic Resonance Imaging) or CT (ComputerTomography).

In certain embodiments, tumor weight increase or tumor growth may beinhibited at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, ormore. Tumor weight or tumor growth may be assessed using techniquesknown to the skilled person.

The detection of tumor growth or the detection of the proliferation oftumor cells may be assessed in vivo by measuring changes in glucoseutilization by positron emission tomography with the glucose analogue2-[18F]-fluor-2-deoxy-D-glucose (FDG-PET) or[18F]-3-fluoro-′3-deoxy-L-thymidine PET. An ex vivo alternative may bestaining of a tumor biopsy with Ki67.

A delay in occurrence of metastases and/or of tumor cell migration maybe a delay of at least one week, one month, several months, one year orlonger. The presence of metastases may be assessed using MRI, CT orEchography or techniques allowing the detection of circulating tumourcells (CTC). Examples of the latter tests are CellSearch CTC test(Veridex), an EpCam-based magnetic sorting of CTCs from peripheralblood. In certain embodiments, tumor growth may be delayed at least oneweek, one month, two months or more. In a certain embodiment, anoccurrence of metastases is delayed at least one week, two weeks, threeweeks, fours weeks, one months, two months, three months, four months,five months, six months or more. A miRNA molecule or an equivalent or amimic or an isomiR thereof or a source thereof wherein a source of saidmiRNA molecule or equivalent or mimic or isomiR thereof comprises atleast 80 nucleotides and comprises a motif having at least 98% identitywith the motif represented by SEQ ID NO:1 had been surprisingly found todelay the occurrence of metastases and/or tumor cell migration in a morepronounced fashion than the corresponding effect of miRNA molecule ofthe 200 family that were already known to have such an effect as shownin Example 2, FIG. 6D. We therefore may anticipate that a miRNA moleculeor a source thereof as identified herein may be considered as anattractive molecule for a use as identified herein.

An increase in the capacity of differentiation of tumor cells may beassessed using a specific differentiation marker and following thepresence of such marker on cells treated. Preferred markers orparameters have already been identified herein, i.e. markers associatedwith MET. This may be done using RT-PCR, western blotting orimmunohistochemistry. An increase of the capacity of differentiation maybe at least a detectable increase after at least one week of treatmentusing any of the identified techniques. Preferably, the increase is of1%, 5%, 10%, 15%, 20%, 25%, or more, which means that the number ofdifferentiated cells within a given sample will increase accordingly. Incertain embodiments, tumor growth may be delayed at least one week, onemonth, two months or more. In a certain embodiment, an occurrence ofmetastases is delayed at least one week, two weeks, three weeks, foursweeks, one months, two months, three months, four months, five months,six months or more.

In a further preferred embodiment, there is provided a compositionfurther comprising another miRNA molecule selected from:

-   -   a) at least one of miRNA-124-1, miRNA-206, miRNA-181a-1,        miRNA-141, miRNA-200a, miRNA-200b, miRNA-200c, miRNA-429 and        miRNA-205 and/or an equivalent or a mimic or an isomiR or a        source thereof.

Since not each of the identified miRNAs molecules or equivalents orisomiRs or mimics thereof is expected to have the same target genes, itis assumed that the use of a miRNA molecule or an equivalent or anisomiR or a mimic thereof wherein a source of said miRNA molecule orequivalent or isomiR or mimic thereof comprises at least 80 nucleotidesand comprises a motif having at least 98% identity with the motifrepresented by SEQ ID NO:1 optionally combined with at least one of themiRNAs molecule, or equivalent or isomiR or mimic thereof or sourcethereof identified above under a) allows a more effective treatment of adisease or condition associated with EMT. Preferred miRNA molecules orequivalents or mimic or isomiR or sources thereof have already beendefined herein. A tumor treated by a composition or a cocktail of atleast a miRNA molecule or an equivalent or an isomiR or a mimic thereofwherein a source of said miRNA molecule or equivalent thereof comprisesat least 80 nucleotides and comprises a motif having at least 98%identity with the motif represented by SEQ ID NO: 1 is expected to havefewer possibilities to escape or to resist said treatment. In a furtherpreferred embodiment, it is encompassed to diagnose the expression ofeach of the miRNA molecules or of their target genes as identifiedherein and depending on the outcome to adapt the identity of the miRNAmolecules used for the treatment.

When the invention relates to a composition comprising more than onemiRNA molecule or equivalent or isomiR or mimic thereof or sourcethereof, it is encompassed that each miRNA molecule or equivalent orisomiR or mimic thereof or source thereof may be present each in aseparate composition, each composition being sequentially orsimulatenously administered to a subject. Alternatively, it is alsoencompassed that more than one miRNA molecules or equivalents or isomiRsor mimics thereof or sources thereof is present in a composition asdefined herein.

In a further aspect, there is provided the use of a miRNA molecule or anequivalent or an isomiR or a mimic thereof wherein a source of saidmiRNA molecule or equivalent or isomiR or mimic thereof comprises atleast 80 nucleotides and comprises a motif having at least 98% identitywith the motif represented by SEQ ID NO:1 or a source thereof or acomposition comprising said miRNA molecule, an equivalent or isomiR ormimic or a source thereof for the manufacture of a medicament forpreventing, treating, reverting, curing and/or delaying a disease or acondition associated with EMT. Each feature of this further aspect hasalready been described herein.

In a further aspect, there is provided a method for preventingpreventing, treating, reverting, curing and/or delaying a condition ordisease associated with EMT by administering a miRNA molecule orequivalent or isomiR or mimic thereof or source thereof or compositionas earlier defined herein to a subject in the need thereof. Each featureof this further aspect has already been described herein.

In a further aspect, there is provided a method for diagnosing EMT or adisease or condition associated with EMT in a subject, the methodcomprising the steps of:

-   -   (a) determining the expression level of a miRNA molecule or an        equivalent or isomiR or mimic thereof wherein a source of said        miRNA molecule or equivalent or isomiR or mimic thereof        comprises at least 80 nucleotides and comprises a motif having        at least 98% identity with the motif represented by SEQ ID NO:1        or a source thereof in a subject, and optionally    -   (b) comparing the expression level of said molecule or        equivalent thereof or source thereof as defined in (a) with a        reference value for the expression level of said molecule,        equivalent, isomiR, mimic or source thereof, the reference value        preferably being the average value for the expression level of        said molecule, equivalent, isomiR, mimic or source thereof in a        healthy subject.

In the context of the invention, diagnosis means either a predictiverisk assessment of a subject for developing a disease or a conditionassociated EMT or for developing EMT itself. In the context of theinvention, a subject may be an animal. Preferably a subject is a mammal.Preferred mammal is a human being. Preferably, a subject is a humanbeing.

Since the expression levels of these nucleotide sequences and/or amountsof corresponding miRNA molecule or equivalent or isomiR or mimic thereofor source thereof may be difficult to be measured in a subject, a samplefrom a subject is preferably used. According to another preferredembodiment, the expression level (of a nucleotide sequence or miRNAmolecule or equivalent or isomiR or mimic or source thereof) isdetermined ex vivo in a sample obtained from a subject. The samplepreferably comprises a body fluid of a subject. A body fluid maycomprise or be derived from blood, serum, plasma, stool, urine or atissue biopsy or a tumor biopsy or a cancer tissue of epithelial originof a subject. Preferred tissue is bladder or prostate. It isspecifically contemplated that the invention can be used to evaluate ordiagnose differences between stages of disease or condition associatedwith EMT, such as between pre-cancer and cancer, or between a primarytumor and a metastasized tumor.

An increase or decrease of the expression level of a nucleotide sequence(or steady state level of the encoded miRNA molecule or equivalent orisomiR or mimic or source thereof) is preferably defined as being adetectable change of the expression level of a nucleotide (or steadystate level of an encoded miRNA molecule or equivalent or isomiR ormimic or source thereof or any detectable change in a biologicalactivity of a miRNA molecule or equivalent or isomiR or mimic or sourcethereof) using a method as defined earlier on as compared to theexpression level of a corresponding nucleotide sequence (or steady statelevel of a corresponding encoded miRNA molecule or equivalent or isomiRor mimic or source thereof) in a healthy subject. A preferred nucleotidesequence is a sequence encoding a precursor of a miRNA molecule orequivalent or isomiR or mimic thereof or a precursor sequence of a miRNAmolecule or equivalent or isomiR or mimic thereof. According to apreferred embodiment, an increase or decrease of a miRNA activity isquantified using a specific assay for a miRNA activity. A preferredassay is the assessment of MET as earlier defined herein.

Preferably, a decrease of the expression level of a nucleotide sequencemeans a decrease of at least 5% of the expression level of thenucleotide sequence using arrays. More preferably, a decrease of theexpression level of a nucleotide sequence means an decrease of at least10%, even more preferably at least 20%, at least 30%, at least 40%, atleast 50%, at least 70%, at least 90%, or 100%. In this case, there isno detectable expression.

Preferably, a decrease of the expression level of a miRNA molecule orequivalent or isomiR or mimic or source thereof means a decrease of atleast 5% of the expression level of the miRNA using qPCR, microarrays orNorthernblot analysis. Preferably qPCR is stem-loop RT qPCR. Morepreferably, a decrease of the expression level of a miRNA molecule orequivalent or isomiR or mimic or source thereof means a decrease of atleast 10%, even more preferably at least 20%, at least 30%, at least40%, at least 50%, at least 70%, at least 90%, or 100%. In this case,there is no detectable expression.

Preferably, a decrease of a miRNA activity means a decrease of at least5% of a miRNA activity using a suitable assay. More preferably, adecrease of a miRNA activity means a decrease of at least 10%, even morepreferably at least 20%, at least 30%, at least 40%, at least 50%, atleast 70%, at least 90%, or 100%. In this case, there is no detectableexpression.

Preferably, an increase of the expression level of a nucleotide sequencemeans an increase of at least 5% of the expression level of thenucleotide sequence using any of the techniques mentioned herein. Morepreferably, an increase of the expression level of a nucleotide sequencemeans an increase of at least 10%, even more preferably at least 20%, atleast 30%, at least 40%, at least 50%, at least 70%, at least 90%, atleast 150% or more.

Preferably, an increase of the expression level of a miRNA molecule orequivalent or isomiR or mimic or source thereof means an increase of atleast 5% of the expression level of the miRNA molecule or equivalent orisomiR or mimic or source thereof using RT-qPCR, preferably stem-loop RTqPCR. More preferably, an increase of the expression level of a miRNAmolecule or equivalent or isomiR or mimic or source thereof means anincrease of at least 10%, even more preferably at least 20%, at least30%, at least 40%, at least 50%, at least 70%, at least 90%, at least150% or more.

Preferably, an increase of a miRNA activity means an increase of atleast 5% of a miRNA activity using a suitable assay. More preferably, anincrease of a miRNA activity means an increase of at least 10%, evenmore preferably at least 20%, at least 30%, at least 40%, at least 50%,at least 70%, at least 90%, at least 150% or more.

Preferably, an expression level is determined ex vivo in a sampleobtained from a subject. More preferably, the sample is as earlierdefined herein and wherein subsequently, a given nucleotide sequenceand/or miRNA molecule or equivalent or isomiR or mimic or source thereofis extracted and purified using known methods to the skilled person.More preferably, the sample is or comprises or is derived from a tumorbiopsy, blood or urine.

In a diagnostic method of the invention preferably the expression levelof more than one, more preferably of at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 miRNAs molecule or equivalent or isomiR or mimicor source thereof and/or the steady state levels of the correspondingmiRNAs molecule or equivalent or isomiR or mimic or source thereof aredetermined.

Accordingly in a preferred method, in step (a) one determines theexpression level of another miRNA molecule or equivalent or sourcethereof selected from:

-   -   (a) at least one of miRNA-124-1, miRNA-206, miRNA-181a-1,        miRNA-141, miRNA-200a, miRNA-200b miRNA-200c, miRNA-429 and        miRNA-205 and/or an equivalent or a source thereof.

In a further preferred method, EMT or a disease or condition associatedwith EMT is diagnosed when the comparison leads to the finding of adecrease of the expression level of said miRNA molecule or an equivalentor a mimic or an isomiR thereof wherein a source of said miRNA moleculeor equivalent thereof comprises at least 80 nucleotides and comprises amotif having at least 98% identity with the motif represented by SEQ IDNO:1. More preferred miRNA molecules or an equivalents or mimics orisomiRs or sources thereof have all been defined earlier herein.

In a further preferred method, EMT or a disease or condition associatedwith EMT is diagnosed when the comparison leads to the finding of adecrease of the expression level of miRNA molecule or an equivalentthereof wherein a source of said miRNA molecule or equivalent thereofcomprises at least 80 nucleotides and comprises a motif having at least98% identity with the motif represented by SEQ ID NO:1 or a sourcethereof and a decrease of the expression level of at least one ofanother miRNA selected from:

(a) at least one of miRNA-124-1, miRNA-206, miRNA-181a-1, miRNA-141,miRNA-200a, miRNA-200b, miRNA-200c, miRNA-429 and miRNA-205 and/or anequivalent or a mimic or an isomiR or a source thereof.

In a further aspect, there is provided a method for identification of asubstance or a molecule capable of preventing, treating, reverting,curing and/or delaying EMT or a condition or disease associated with EMTin a subject, the method comprising the steps of:

-   -   (a) providing a test cell population capable of expressing a        miRNA molecule or an equivalent or a mimic or an isomiR thereof,        wherein a source of said miRNA molecule or equivalent or mimic        or isomiR thereof comprises at least 80 nucleotides and        comprises a motif having at least 98% identity with the motif        represented by SEQ ID NO:1 or source thereof, preferably the        test population comprises bladder or prostate cells, and/or the        test cell population comprises cancer cells and/or the test cell        population comprises mammalian cells, and/or the test cell        population comprises human cells;

(b) contacting the test cell population with the substance;

-   -   (c) determining the expression level of said miRNA molecule or        equivalent or mimic or isomiR thereof or source thereof or the        activity or steady state level of said miRNA molecule or        equivalent or mimic or isomiR thereof or source thereof in the        test cell population contacted with the substance;    -   (d) comparing the expression, activity or steady state level        determined in (c) with the expression, activity or steady state        level of said miRNA molecule or equivalent or mimic or isomiR        thereof or source thereof in a test cell population that is not        contacted with the substance; and,    -   (e) identifying a substance that produces a difference in        expression level, activity or steady state level of said miRNA        molecule or equivalent or mimic or isomiR thereof or source        thereof, between the test cell population that is contacted with        the substance and the test cell population that is not contacted        with the substance.

Preferably, in step a), a test cell comprises a nucleic acid constructcomprising a source or a precursor of a miRNA molecule or an equivalentor a mimic or an isomiR thereof wherein a source or a precursor of saidmiRNA molecule or equivalent or mimic or isomiR thereof comprises atleast 80 nucleotides and comprises a motif having at least 98% identitywith the motif represented by SEQ ID NO:1. More preferred miRNAmolecules or an equivalents or mimics or isomiRs or sources thereof haveall been defined earlier herein. More preferably, a test cell comprisesa CDH1 promotor driven firefly luciferase construct. Preferably, in amethod the expression levels, an activity or steady state levels of morethan one nucleotide sequence or more than one miRNA molecule, equivalentor mimic or isomiR or source thereof are compared. Preferably, in amethod, a test cell population comprises mammalian cells, morepreferably human cells. Even more preferably, a test cell populationcomprises bladder or prostate cells. A preferred test cell populationdoes not express a miRNA molecule or an equivalent or a mimic or anisomiR thereof wherein a source of said miRNA molecule or equivalentthereof comprises at least 80 nucleotides and comprises a motif havingat least 98% identity with the motif represented by SEQ ID NO:1 orsource thereof or has a reduced expression compared to a normalepithelial counterpart that expresses CDH1. More preferably, a test cellpopulation comprises a mesenchymal cell with low CDH1 expression, but iscapable of expressing CDH1. Alternatively or in addition to previousmentioned cells, in one aspect the invention also pertains to asubstance that is identified in the aforementioned methods.

In a preferred method, the expression levels, activities or steady statelevels of at least another one miRNA molecule or equivalent or mimic orisomiR or source thereof is compared, preferably wherein the other miRNAmolecule or equivalent or mimic or isomiR or source thereof is selectedfrom:

(a) at least one of miRNA-124-1, miRNA-206, miRNA-181a-1, miRNA-141,miRNA-200a, miRNA-200b, miRNA-200c, miRNA-429 and miRNA-205 and/or anequivalent, a mimic or an isomiR or a source thereof.

General Definitions and General Technologies Referred to Herein

MicroRNA molecules (“miRNAs”) are generally 21 to 22 nucleotides inlength, though lengths of 17 and up to 25 nucleotides have beenreported. Any length of 17, 18, 19, 20, 21, 22, 23, 24, 25 is thereforeencompassed within the present invention. The miRNAs are each processedfrom a longer precursor RNA molecule (“precursor miRNA”). PrecursormiRNAs are transcribed from non-protein-encoding genes. A precursor mayhave a length of at least 50, 70, 75, 80, 85, 100, 150, 200 nucleotidesor more. The precursor miRNAs have two regions of complementarity thatenables them to form a stem-loop- or fold-back-like structure, which iscleaved by enzymes called Dicer and Drosha in animals. Dicer and Droshaare ribonuclease Ill-like nucleases. The processed miRNA is typically aportion of the stem.

The processed miRNA (also referred to as “mature miRNA”) becomes part ofa large complex, known as the RNA-Induced Silencing Complex (RISC)complex, to (down)-regulate a particular target gene. Examples of animalmiRNAs include those that perfectly or imperfectly basepair with themRNA target, resulting in either mRNA degradation or inhibition oftranslation respectively (Olsen et al, 1999; Seggerson et al, 2002).SiRNA molecules also are processed by Dicer, but from a long,double-stranded RNA molecule. SiRNAs are not naturally found in animalcells, but they can function in such cells in a RNA-induced silencingcomplex (RISC) to direct the sequence-specific cleavage of an mRNAtarget (Denli et al, 2003).

The study of endogenous miRNA molecules is described in U.S. PatentApplication 60/575,743, which is hereby incorporated by reference in itsentirety. A miRNA is apparently active in the cell when the mature,single-stranded RNA is bound by a protein complex that regulates thetranslation of mRNAs that hybridize to the miRNA. Introducing exogenousRNA molecules that affect cells in the same way as endogenouslyexpressed miRNAs requires that a single-stranded RNA molecule of thesame sequence as the endogenous mature miRNA be taken up by the proteincomplex that facilitates translational control. A variety of RNAmolecule designs have been evaluated. Three general designs thatmaximize uptake of the desired single-stranded miRNA by the miRNApathway have been identified. An RNA molecule with a miRNA sequencehaving at least one of the three designs may be referred to as asynthetic miRNA.

miRNA molecules of the invention can replace or supplement the genesilencing activity of an endogenous miRNA. An example of such molecules,preferred characteristics and modifications of such molecules andcompositions comprising such molecules is described in WO2009/091982,which is hereby incorporated by reference in its entirety.

miRNA molecules of the invention or equivalents or source thereofcomprise, in some embodiments, two RNA molecules wherein one RNA isidentical to a naturally occurring, mature miRNA. The RNA molecule thatis identical to a mature miRNA is referred to as the active strand. Thesecond RNA molecule, referred to as the complementary strand, is atleast partially complementary to the active strand. The active andcomplementary strands are hybridized to create a double-stranded RNA,that is similar to the naturally occurring miRNA precursor that is boundby the protein complex immediately prior to miRNA activation in thecell. Maximizing activity of said miRNA requires maximizing uptake ofthe active strand and minimizing uptake of the complementary strand bythe miRNA protein complex that regulates gene expression at the level oftranslation. The molecular designs that provide optimal miRNA activityinvolve modifications of the complementary strand.

Two designs incorporate chemical modifications of the complementarystrand.

The first modification involves creating a complementary RNA with agroup other than a phosphate or hydroxyl at its 5′ terminus. Thepresence of the 5′ modification apparently eliminates uptake of thecomplementary strand and subsequently favors uptake of the active strandby the miRNA protein complex. The 5′ modification can be any of avariety of molecules including NH2, NHCOCH3, biotin, and others.

The second chemical modification strategy that significantly reducesuptake of the complementary strand by the miRNA pathway is incorporatingnucleotides with sugar modifications in the first 2-6 nucleotides of thecomplementary strand. It should be noted that the sugar modificationsconsistent with the second design strategy can be coupled with 5′terminal modifications consistent with the first design strategy tofurther enhance miRNA activities.

The third miRNA design involves incorporating nucleotides in the 3′ endof the complementary strand that are not complementary to the activestrand.

Hybrids of the resulting active and complementary RNAs are very stableat the 3′ end of the active strand but relatively unstable at the 5′ endof the active strand. Studies with siRNAs indicate that 5′ hybridstability is a key indicator of RNA uptake by the protein complex thatsupports RNA interference, which is at least related to the miRNApathway in cells. The inventors have found that the judicious use ofmismatches in the complementary RNA strand significantly enhances theactivity of said miRNA.

MiRNA Libraries

A key application for the miRNAs as identified herein is the assessmentor diagnosis of the presence of one individual or groups of miRNAs in asample. Cell populations with each of the different miRNAs can then beassayed to identify miRNAs whose presence affects a cellular phenotype(i.e. EMT). The number of different miRNAs in the libraries is variable.It is contemplated that there may be, be at least, or be at most 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more,or any range derivable therein, different miRNA-specific molecules inthe library. In specific embodiments, libraries have 1 to 20 differentmiRNA-specific molecules, or 5 to 20 different miRNA-specific molecules.“Different” miRNA-specific molecules refers to nucleic acids thatspecifically encode miRNAs with different sequences.

miRNAs are contemplated to be made primarily of RNA, though in someembodiments, they may be RNA, nucleotide analogs, such as Locked nucleicacids (LNA) or Unlocked nucleic acids (UNA), DNA, or any combination ofDNA, RNA, nucleotide analogs, and PNAs (Peptide Nucleic Acids).Accordingly, it is understood that the library contains one or morenucleic acids for these different miRNAs. In specific embodiments, thelibrary is specific to human miRNAs, though libraries for multipleorganisms are contemplated.

An RNA molecule of the invention has or comprises or consists of a miRNAregion. In specific embodiments, a miRNA molecule or equivalent thereofhas a sequence that derives from any of SEQ ID NOs: 2-21 inclusive(Table 3). It is particularly contemplated that nucleic acid moleculesof the invention may be derived from any of the mature miRNA sequencesin SEQ ID NOs: 2-21.

A miRNA molecule or equivalent thereof will include a sequence thatextends at least 1 to 5 nucleotides of coding sequence upstream and/ordownstream of the predicted miRNA sequence. In some embodiments,molecules have up to 1, 2, 3, 4, 5, 6, 7, or more contiguousnucleotides, or any range derivable therein, that flank the sequenceencoding the predominant processed miRNA on one or both sides (5′ and/or3′ end).

Libraries of the invention can contain miRNA sequences from any organismhaving miRNAs, specifically including but not limited to, mammals suchas humans, non human primates, rats and mice. Specifically contemplatedare libraries having, having at least, or having at most 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more differentmiRNAs (that is, miRNA-specific molecules having different sequencesderived from different miRNA genes). Specifically contemplated are suchlibraries described in the previous sentence with respect to any of SEQID NOs: 2-21 particularly those corresponding to miRNA sequences (maturesequence).

Nucleic Acids

The present invention concerns nucleic acid molecules also calledsources or precursors of miRNAs that can introduce miRNAs in culturedcells or into a subject. The nucleic acids may have been produced incells or in vitro by purified enzymes though they are preferentiallyproduced by chemical synthesis. They may be crude or purified. The term“miRNA,” unless otherwise indicated, refers to the processed miRNA,after it has been cleaved from its precursor. Table 2 indicates whichSEQ ID NO corresponds to a particular precursor sequence of a miRNA (SEQID NO: 22-35 and Table 3 which SEQ ID NO corresponds to the maturesequence of a miRNA (SEQ ID NO: 2-21. Table 4 identifies the cloned DNAsequences into the lentiviral vector (SEQ ID NO: 36-47, which were usedin the functional screen as described in the examples. Table 5identifies the preferred seed sequence (as SEQ ID NO: 87-107) of each ofthe mature miRNAs of Table 3. The name of the miRNA is often abbreviatedand referred to without the prefix and will be understood as such,depending on the context. Unless otherwise indicated, miRNAs referred toin the application are human sequences identified as mir-X or let-X,where X is a number and/or letter.

It is understood that a miRNA is derived from genomic sequences or anon-coding gene. In this respect, the term “gene” is used for simplicityto refer to the genomic sequence encoding the precursor miRNA for agiven miRNA. However, embodiments of the invention may involve genomicsequences of a miRNA that are involved in its expression, such as apromoter or other regulatory sequences.

The term “recombinant” may be used and this generally refers to amolecule that has been manipulated in vitro or that is the replicated orexpressed product of such a molecule.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (one or more strands) ofDNA, RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompasses the terms “oligonucleotide”and “polynucleotide,” each as a subgenus of the term “nucleic acid.”

The term “miRNA” generally refers to a single-stranded molecule, but inspecific embodiments, molecules implemented in the invention will alsoencompass a region or an additional strand that is partially (between 10and 50% complementary across length of strand), substantially (greaterthan 50% but less than 100% complementary across length of strand) orfully complementary to another region of the same single-strandedmolecule or to another nucleic acid. Thus, nucleic acids may encompass amolecule that comprises one or more complementary or self-complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. For example, precursor miRNA may have a self-complementaryregion, which is up to 100% complementary.

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature using techniques known to the skilled person such as southernblotting procedures. The term “anneal” as used herein is synonymous with“hybridize.” The term “hybridization”, “hybridize(s)” or “capable ofhybridizing” may mean “low”, “medium” or “high” hybridization conditionsas defined below.

Low to medium to high stringency conditions means prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 pg/ml sheared anddenatured salmon sperm DNA, and either 25% 35% or 50% formamide for lowto medium to high stringencies respectively. Subsequently, thehybridization reaction is washed three times for 30 minutes each using2×SSC, 0.2% SDS and either 55° C., 65° C., or 75° C. for low to mediumto high stringencies.

Nucleic acids or derivaties thereof of the invention will comprise, insome embodiments the miRNA sequence of any miRNA described in SEQ IDNOs: 2-21 or are described in SEQ ID NO: 22-35 or in SEQ ID NO: 36-47.It is contemplated that nucleic acids sequences of the invention derivedfrom SEQ ID NO: 2-21 can have, have at least, or have at most 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, contiguousnucleotides from SEQ ID NOs: 2-21 (or any range derivable therein). Inother embodiments, nucleic acids are, are at least, or are at most 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100% identical to the miRNA sequence of SEQ ID NOs: 2-21 or to theprecursor sequence of any of SEQ ID NO: 22-35 or any combination orrange derivable therein.

Nucleobases

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in a manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moeity. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.)moieties comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like. Other examplesare well known to those of skill in the art.

A nucleobase may be comprised in a nucleoside or nucleotide, using anychemical or natural synthesis method described herein or known to one ofordinary skill in the art. Such nucleobase may be labeled or it may bepart of a molecule that is labeled and contains the nucleobase.

Nucleosides

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992).

Nucleotides

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

Nucleic Acid Analogs

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. RNAwith nucleic acid analogs may also be labeled according to methods ofthe invention. As used herein a “derivative” refers to a chemicallymodified or altered form of a naturally occurring molecule, while theterms “mimic” or “analog” refer to a molecule that may or may notstructurally resemble a naturally occurring molecule or moiety, butpossesses similar functions. As used herein, a “moiety” generally refersto a smaller chemical or molecular component of a larger chemical ormolecular structure. Nucleobase, nucleoside and nucleotide analogs orderivatives are well known in the art, and have been described (see forexample, Scheit, 1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in: U.S. Pat. No. 5,681,947, which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167, which describe nucleic acids incorporating fluorescentanalogs of nucleosides found in DNA or RNA, particularly for use asfluorescent nucleic acids probes; U.S. Pat. No. 5,614,617, whichdescribes oligonucleotide analogs with substitutions on pyrimidine ringsthat possess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663,5,872,232 and 5,859,221, which describe oligonucleotide analogs withmodified 5-carbon sugars (i.e., modified T-deoxyfuranosyl moieties) usedin nucleic acid detection; U.S. Pat. No. 5,446,137, which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165, whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606, which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697, which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847, which describe the linkage of asubstituent moiety which may comprise a drug or label to the 2′ carbonof an oligonucleotide to provide enhanced nuclease stability and abilityto deliver drugs or detection moieties; U.S. Pat. No. 5,223,618, whichdescribes oligonucleotide analogs with a 2′ or 3′ carbon backbonelinkage attaching the 4′ position and 3′ position of adjacent 5-carbonsugar moiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967, which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240, which describe oligonucleotides with three or four atomlinker moiety replacing phosphodiester backbone moiety used for improvednuclease resistance, cellular uptake and regulating RNA expression; U.S.Pat. No. 5,858,988, which describes hydrophobic carrier agent attachedto the 2′-0 position of oligonucleotides to enhanced their membranepermeability and stability; U.S. Pat. No. 5,214,136, which describesoligonucleotides conjugated to anthraquinone at the 5′ terminus thatpossess enhanced hybridization to DNA or RNA; enhanced stability tonucleases; U.S. Pat. No. 5,700,922, which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythro-pentofuranosyl nucleotidesfor enhanced nuclease resistance, binding affinity, and ability toactivate RNase H; and WO98/39352, WO99/14226, WO2003/95467 andWO2007/085485, which describe modified RNA nucleotides of which theribose moiety is modified with an extra bridge connecting the 2′ oxygenand 4′ carbon. The locked ribose significantly increases the bindingaffinity and specificity; and WO2008/147824, which describes modifiedRNA nucleotides termed UNA (unlocked nucleic acid). UNA are acyclicanalogues of RNA in which the bond between the C2′ and C3′ atoms hasbeen cleaved, decreasing binding affinity towards a complementarystrand. UNA are compatible with RNase H recognition and RNA cleavage andimproves siRNA mediated gene silencing; WO2008/036127 which describesMorpholino nucleic acid analogues, which contain both uncharged andcationic intersubunit linkages; WO/2007/069092 and EP2075342 whichdescribe Zip Nucleic Acids (ZNA), containing conjugating sperminederivatives as cationic moieties (Z units) to an oligonucleotide; U.S.Pat. No. 5,708,154, which describes RNA linked to a DNA to form aDNA-RNA hybrid; U.S. Pat. No. 5,728,525, which describes the labeling ofnucleoside analogs with a universal fluorescent label.

Additional teachings for nucleoside analogs and nucleic acid analogs areU.S. Pat. No. 5,728,525, which describes nucleoside analogs that areend-labeled; U.S. Pat. No. 5,637,683, 6,251,666 (L-nucleotidesubstitutions), and U.S. Pat. No. 5,480,980 (7-deaza-2′-deoxyguanosinenucleotides and nucleic acid analogs thereof).

The use of other analogs is specifically contemplated for use in thecontext of the present invention. Such analogs may be used in syntheticnucleic acid molecules of the invention, both throughout the molecule orat selected nucleotides. They include, but are not limited to,

1) ribose modifications (such as 2′F, 2′ NH2, 2′N3,4′thio, or 2′ O—CH3)and2) phosphate modifications (such as those found in phosphorothioates,methyl phosphonates, and phosphoroborates).

Such analogs have been created to confer stability on RNAs by reducingor eliminating their capacity to be cleaved by ribonucleases. When thesenucleotide analogs are present in RNAs, they can have profoundlypositive effects on the stability of the RNAs in animals. It iscontemplated that the use of nucleotide analogs can be used alone or inconjunction with any of the design modifications of a synthetic miRNAfor any nucleic acid of the invention.

Modified Nucleotides

miRNAs of the invention specifically contemplate the use of nucleotidesthat are modified to enhance their activities. Such nucleotides includethose that are at the 5′ or 3′ terminus of the RNA as well as those thatare internal within the molecule. Modified nucleotides used in thecomplementary strands of said miRNAs either block the 5′ OH or phosphateof the RNA or introduce internal sugar modifications that enhance uptakeof the active strand of the miRNA. Modifications for the miRNAs includeinternal sugar modifications that enhance hybridization as well asstabilize the molecules in cells and terminal modifications that furtherstabilize the nucleic acids in cells. Further contemplated aremodifications that can be detected by microscopy or other methods toidentify cells that contain the synthetic miRNAs.

Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Though miRNAs according to theinvention could be produced using recombinant methods, it is preferredto produce miRNAs by chemical synthesis or enzymatic production. miRNAscan be produced by a number of methods, including methods involvingrecombinant DNA technology.

Nucleic acid synthesis is performed according to standard methods. See,for example, Itakura and Riggs (1980). Additionally, U.S. Pat. No.4,704,362, U.S. Pat. No. 5,221,619, and U.S. Pat. No. 5,583,013 eachdescribe various methods of preparing nucleic acids. Non-limitingexamples of a nucleic acid (e.g., a oligonucleotide), include a nucleicacid made by in vitro chemically synthesis using phosphotriester,phosphite or phosphoramidite chemistry and solid phase techniques suchas described in EP 266,032, incorporated herein by reference, or viadeoxynucleoside H-phosphonate intermediates as described by Froehler etal., 1986 and U.S. Pat. No. 5,705,629, each incorporated herein byreference. In the methods of the present invention, one or moreoligonucleotide may be used. Various different mechanisms ofoligonucleotide synthesis have been disclosed in for example, U.S. Pat.Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148,5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein byreference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference.

Oligonucleotide synthesis is well known to those of skill in the art.Various different mechanisms of oligonucleotide synthesis have beendisclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,5,602,244, each of which is incorporated herein by reference.

Basically, chemical synthesis can be achieved by the diester method, thetriester method polynucleotides phosphorylase method and by solid-phasechemistry. These methods are discussed in further detail below.

Diester Method

The diester method was the first to be developed to a usable state,primarily by Khorana and co-workers. (Khorana, 1979). The basic step isthe joining of two suitably protected deoxynucleotides to form adideoxynucleotide containing a phosphodiester bond. The diester methodis well established and has been used to synthesize DNA molecules(Khorana, 1979).

Triester Method

The main difference between the diester and triester methods is thepresence in the latter of an extra protecting group on the phosphateatoms of the reactants and products (Itakura et al., 1975). Thephosphate protecting group is usually a chlorophenyl group, whichrenders the nucleotides and polynucleotide intermediates soluble inorganic solvents. Therefore purifications are done in chloroformsolutions. Other improvements in the method include (i) the blockcoupling of trimers and larger oligomers, (ii) the extensive use ofhigh-performance liquid chromatography for the purification of bothintermediate and final products, and (iii) solid-phase synthesis.

Polynucleotide Phosphorylase Method.

This is an enzymatic method of DNA synthesis that can be used tosynthesize many useful oligonucleotides (Gillam et al., 1978; Gillam etal, 1979). Under controlled conditions, polynucleotide phosphorylaseadds predominantly a single nucleotide to a short oligonucleotide.

Chromatographic purification allows the desired single adduct to beobtained. At least a trimer is required to start the procedure, and thisprimer must be obtained by some other method. The polynucleotidephosphorylase method works and has the advantage that the proceduresinvolved are familiar to most biochemists.

Solid-Phase Methods.

Drawing on the technology developed for the solid-phase synthesis ofpolypeptides, it has been possible to attach the initial nucleotide tosolid support material and proceed with the stepwise addition ofnucleotides. All mixing and washing steps are simplified, and theprocedure becomes amenable to automation. These syntheses are nowroutinely carried out using automatic nucleic acid synthesizers.

Phosphoramidite chemistry (Beaucage and Lyer, 1992) has become by farthe most widely used coupling chemistry for the synthesis ofoligonucleotides. As is well known to those skilled in the art,phosphoramidite synthesis of oligonucleotides involves activation ofnucleoside phosphoramidite monomer precursors by reaction with anactivating agent to form activated intermediates, followed by sequentialaddition of the activated intermediates to the growing oligonucleotidechain (generally anchored at one end to a suitable solid support) toform the oligonucleotide product.

Recombinant Methods.

Recombinant methods for producing nucleic acids in a cell are well knownto those of skill in the art. These include the use of vectors,plasmids, cosmids, and other vehicles for delivery a nucleic acid to acell, which may be the target cell or simply a host cell (to producelarge quantities of the desired RNA molecule). Alternatively, suchvehicles can be used in the context of a cell free system so long as thereagents for generating the RNA molecule are present. Such methodsinclude those described in Sambrook, 2003, Sambrook, 2001 and Sambrook,1989, which are hereby incorporated by reference. In certainembodiments, the present invention concerns nucleic acid molecules thatare not synthetic. In some embodiments, the nucleic acid molecule has achemical structure of a naturally occuring nucleic acid and a sequenceof a naturally occuring nucleic acid, such as the exact and entiresequence of a single stranded primary miRNA (see Lee 2002), asingle-stranded precursor miRNA, or a single-stranded mature miRNA. Inaddition to the use of recombinant technology, such non-syntheticnucleic acids may be generated chemically, such as by employingtechnology used for creating oligonucleotides.

Design of miRNAs

miRNAs typically comprise two strands, an active strand that isidentical in sequence to the mature miRNA that is being studied and acomplementary strand that is at least partially complementary to theactive strand. The active strand is the biologically relevant moleculeand should be preferentially taken up by the complex in cells thatmodulates translation either through mRNA degradation or translationalcontrol. Preferential uptake of the active strand has two profoundresults: (1) the observed activity of said miRNA increases dramaticallyand (2) non-intended effects induced by uptake and activation of thecomplementary strand are essentially eliminated. According to theinvention, several miRNA designs can be used to ensure the preferentialuptake of the active strand.

5′ Blocking Agent.

The introduction of a stable moiety other than phosphate or hydroxyl atthe 5′ end of the complementary strand impairs its activity in the miRNApathway. This ensures that only the active strand of the miRNA will beused to regulate translation in the cell. 5′ modifications include, butare not limited to, NH2, biotin, an amine group, a lower alkylaminegroup, an acetyl group, 2′ 0-Me, DMTO, fluoroscein, a thiol, or acridineor any other group with this type of functionality.

Other sense strand modifications. The introduction of nucleotidemodifications like 2′-O Me, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA), NH2, biotin, an amine group, a loweralkylamine group, an acetyl group, DMTO, fluoroscein, a thiol, oracridine or any other group with this type of functionality in thecomplementary strand of the miRNA can eliminate the activity of thecomplementary strand and enhance uptake of the active strand of themiRNA.

Base mismatches in the sense strand. As with siRNAs (Schwarz 2003), therelative stability of the 5′ and 3′ ends of the active strand of themiRNA apparently determines the uptake and activation of the active bythe miRNA pathway. Destabilizing the 5′ end of the active strand of themiRNA by the strategic placement of base mismatches in the 3′ end of thecomplementary strand of the synthetic miRNA enhances the activity of theactive strand and essentially eliminates the activity of thecomplementary strand.

Host Cells and Target Cells

The cells wherein a miRNA or source thereof is introduced or wherein thepresence of a miRNA is assessed may be derived from or contained in anyorganism. Preferably, the cell is a vertebrate cell. More preferably,the cell is a mammalian cell. Even more preferably, the cell is a humancell.

A mammalian cell may be from the germ line or somatic, totipotent orpluripotent, dividing or non-dividing, epithelium, immortalized ortransformed, or the like. The cell may be an undifferentiated cell, suchas a stem cell, or a differentiated cell, such as from a cell of anorgan or tissue. Alternatively, cells may be qualified as epithelialcells, brain, breast, cervix, colon, gastrointestinal tract, heart,kidney, large intestine, liver, lung, ovary, pancreas, heart, prostate,bladder, small intestine, stomach, testes or uterus.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations formed by cell division. Itis understood that all progeny may not be identical due to deliberate orinadvertent mutations. A host cell may be “transfected” or“transformed,” which refers to a process by which exogenous nucleic acidis transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny. As used herein, theterms “engineered” and “recombinant” cells or host cells are intended torefer to a cell into which an exogenous nucleic acid sequence, such as,for example, a small, interfering RNA or a template construct encoding areporter gene has been introduced. Therefore, recombinant cells aredistinguishable from naturally occurring cells that do not contain arecombinantly introduced nucleic acid.

A tissue may comprise a host cell or cells to be transformed orcontacted with a nucleic acid delivery composition and/or an additionalagent. The tissue may be part or separated from an organism. In certainembodiments, a tissue and its constituent cells may comprise, but is notlimited to brain, stem cells, liver, lung, bone, breast, cervix, colon,endometrium, epithelial, esophagus, goblet cells, kidney, ovaries,pancreas, prostate, bladder, skin, small intestine, stomach, testes,heart, blood vessel.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be amammal, a human, a primate or murine. One of skill in the art wouldfurther understand the conditions under which to incubate all of theabove described host cells to maintain them and to permit their divisionto form progeny.

Delivery Methods

The present invention involves in some embodiments delivering a nucleicacid into a cell. This may be done as part of a screening method, or itmay be related to a therapeutic or diagnostic application.

RNA molecules may be encoded by a nucleic acid molecule comprised in avector. The term “vector” is used to refer to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses,lentivirus, and plant viruses), and artificial chromosomes (e.g., YACs).One of skill in the art would be well equipped to construct a vectorthrough standard recombinant techniques, which are described in Sambrooket al, 1989 and Ausubel et al, 1996, both incorporated herein byreference. In addition to encoding a modified polypeptide such asmodified gelonin, a vector may encode non-modified polypeptide sequencessuch as a tag or targetting molecule. A targetting molecule is one thatdirects the desired nucleic acid to a particular organ, tissue, cell, orother location in a subject's body.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described

There are a number of ways in which expression vectors may be introducedinto cells. In certain embodiments of the invention, the expressionvector comprises a virus or engineered vector derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986). The expression vectors may containan RNAi expression cassette comprising one promoter and one or morestem-loop structures separated by one or more spacer regions(WO2006/084209).

Another way of introducing expression vectors into cells, using avidinfusion proteins is described in U.S. Pat. No. 6,287,792.

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells; they can also be used as vectors. Other viral vectorsmay be employed as expression constructs in the present invention.Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus(AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska,1984), lentivirus (WO2008/071959, WO2004/054512), Hemaglutinating Virusof Japan (WO2004/035779), Baculovirus (WO2006/048662) and herpesvirusesmay be employed. They offer several attractive features for variousmammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden,1986; Coupar et al, 1988; Horwich et al, 1990).

Other suitable methods for nucleic acid delivery to affect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al, 1987); by liposome mediated transfection (Nicolau and Sene, 1982;Fraley et al., 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneda etal., 1989; Kato et al., 1991); by photochemical internalization(WO2008/007073); by microprojectile bombardment (PCT Application Nos. WO94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055,5,550,318, 5,538,877 and 5,538,880, and each incorporated herein byreference); by agitation with silicon carbide fibers (Kaeppler et al,1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated hereinby reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos.5,591,616 and 5,563,055, each incorporated herein by reference); or byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S.Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein byreference); by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985). Through the application of techniques such as these,organelle(s), cell(s), tissue(s) or organism(s) may be stably ortransiently transformed.

A review provides several ways of formulating a RNA molecule in order tooptimize its internalisation into a cell (Kim SS., et al, Trends Mol.Med., 2009, 15: 491-500). The following other publications disclosesalternative ways of formulating a RNA molecule in order to improve itsinternalisation into a cell, each incorporated herein by reference: WO2007/095152, describing the use of PTD-DRBD (Peptide transductiondomains linked to double stranded binding domain) for delivery ofoligonculeotides, WO 2009/086558, describing the use of SNALP (StableNucleic Acid Lipid Particles) particles, comprising a mixture ofcationic and fusogenic lipids that enable the cellular uptake andendosomal release of the particle's nucleic acid payload, WO2009/149418, describing neutral phospholipid-oil-RNAi emulsions, WO2007/121947, describing the use of a delivery vehicle based on lipoplex,WO 2009/132131, describing the use of novel lipids and nucleicacid-lipid particles that provide efficient encapsulation and efficientdelivery of the encapsulated nucleic acid to cells, WO2004/091578 andWO2004/064805 describing cochleate technology of alternating layers oflipids that spiral around a nucleic acid molecule, WO2003/047494 andWO2003/047493 describing reverse micelles incorporating nucleic acidsfor oral and mucosal delivery, WO 2008/156702, describing bacteria andbacterial therapeutic particle (BTP), including oligonucleotides for asdelivery vehicle to cells. Each of the formulations referred to ordisclosed in these publications is encompassed by the present invention.

A variety of compounds have been attached to the ends ofoligonucleotides to facilitate their transport across cell membranes.Short signal peptides found in the HIV TAT, HSV VP22, Drosphilaantennapedia, and other proteins have been found to enable the rapidtransfer of biomolecules across membranes (reviewed by Schwarze 2000).These signal peptides, referred to as Protein Transduction Domains(PTDs), have been attached to oligonucleotides to facilitate theirdelivery into cultured cells (Eguchi A, Dowdy S F, Trends PharmacolSci., 2009, 7:341-5). Cholesterols have been conjugated tooligonucleotides to improve their uptake into cells in animals(MacKellar 1992). The terminal cholesterol groups apparently interactwith receptors or lipids on the surfaces of cells and facilitate theinternalization of the modified oligonucleotides. Likewise,poly-L-lysine has been conjugated to oligonucleotides to decrease thenet negative charge and improve uptake into cells (Leonetti 1990).

A variety of compounds have been developed that complex with nucleicacids, deliver them to surfaces of cells, and facilitate their uptake inand release from endosomes. Among these are: (1) a variety of lipidssuch as DOTAP (or other cationic lipid), DDAB, DHDEAB, and DOPE and (2)non-lipid-based polymers like polyethylenimine, polyamidoamine, anddendrimers of these and other polymers. In certain of these embodimentsa combination of lipids is employed such as DOTAP and cholesterol or acholesterol derivative (U.S. Pat. No. 6,770,291, which is herebyincorporated by reference). Several of these reagents have been shown tofacilitate nucleic acid uptake in animals.

The cellular components involved in the miRNA pathway are becomingknown. Proteins that stabilize and/or transport miRNAs within cellsmight enhance the stability and activity of miRNAs because they shouldprotect and guide the bound miRNAs once they are in cells. Mixtures ofmiRNA-transporter proteins and miRNAs could enhance the efficacy ofmiRNA-based therapeutics. RNAs are hydrophilic molecules by virtue oftheir anionic phosphate and sugar backbone. Although the nucleobases arehydrophobic, hydrophilicity dominates owing to the extensive hydrogenbonding resulting from the phosphate and sugar residues. The hydrophiliccharacter and anionic backbone reduces cellular permeation. Conjugationof lipophilic groups like cholesterol (Manoharan, 2002) and lauric andlithocholic acid derivatives with C32 functionality (Lorenz et al,2004), have been shown to improve cellular uptake. Moreover binding ofsteroid conjugated oligonucleotides to different lipoproteins in thebloodstream, such as LDL, protect their integrity and govern theirbiodistribution (Rump et al, 2000). Cholesterol attached to anti-sensemolecules (Bijsterbosch et al., 2001) and aptamers (Rusconi et al.,2004) has also been shown to stabilize oligonucleotides by allowingbinding to lipoproteins. Cholesterol has been demonstrated to enhanceuptake and serum stability of siRNAs in vitro (Lorenz et al., 2004) andin vivo (Soutschek et al., 2004). Additionally, a number of smallmolecules like SB-435495 (Blackie et al, (2002), Isradipine (Oravcova etal, 1994), amlodipine (Oravcova et al, 1994) and2,2′,4,4′,5,5′-hexachlorobiphenyl (Borlakoglu et al, 1990) could enhancecellular uptake, and improve nuclease resistance by promotinglipoprotein association.

Screening with miRNA Libraries

As used in the patent application, screening is a process whereinmultiple miRNA-specific reagents are delivered separately intoindividual cell populations or animals. At one or more designated timesafter delivery, the cell populations or animals are assayed for one ormore phenotypes. Those cells or animals that have a significantlydifferent phenotype than cells or animals in the negative control groupare classified as positives. The miRNA that was being manipulated in thesample is defined as a hit. Hits represent targets for additionalresearch and potential therapeutic development.

In some embodiments, there is a multi-step process for screening, incertain embodiments, there are four general steps:

(1) Develop Quantitative Assay to Monitor Cellular Process beingStudied.

Assays that measure the intensity of a cellular phenotype range frommicroscopic assays that monitor cell size, cell cycle status, orantibody staining to enzymatic assays that assess the turnover of aspecific substrate in a cell lysate to direct measurements ofbiomolecules or small molecules in lysates, on cells, or in medium.

Critical to the success of a screen is creating an assay that trulymeasures the cellular phenotype and maximizing the signal-to-noise ratioof the assay. Maximizing signal-to-noise involves testing variables likeassay time, assay components, cell type, and length of time betweentransfection and assay. The greater the difference in the assay resultsbetween a positive phenotype and a negative control phenotype, thegreater the spread will be in the screening results and the better theopportunity will be to identify interesting genes.

(2) Optimize Transfection Conditions for the Desired Cells.

The first step in this process is identifying a transfection reagent andplating conditions that maximize the uptake of synthetic miRNAs whilemaintaining high cell viability. We find it useful to test 2-5 differenttransfection reagents when using cell lines or 5-10 elelctroporationconditions when using primary or suspension cells. Transfection can beoptimized for the reagent or electroporation condition that worked bestamong the conditions tested. Screening miRNA-specific libraries requiresconditions for high-throughput transfection. In this type of screen,lentiviral introduction rather than transfection was used. This mayrequire alternative optimization techniques.

(3) Screen

Once the assay and transfection process have been developed, a libraryof synthetic miRNAs or miRNAs expressed by viruses can be introducedsequentially into cells in a 24- or 96-well plate. Duplicate ortriplicate transfections for each reagent provide enough data forreasonable statistical analysis.

(4) Validate Hits

Validating a hit involves showing that the observed phenotype is due tothe miRNA being targeted. Hits are typically confirmed by delivering adilution series of the miRNA inhibitor or synthetic miRNA thatregistered as a hit into the cell that was originally assayed.Confirmation is slightly different from validation. Confirmation is arepeat of the miRNA-induced phenotype, whereas validation can alsoinclude reversal of the phenotype by antagonizing miRNA mediatedphenotype.

Labeling and Labeling Techniques

In some embodiments, the present invention concerns miRNAs that arelabeled, such as for screening assays to evaluate the therapeutic ordiagnostic relevance of a particular miRNA species. It is contemplatedthat miRNA may first be isolated (either from a cell in which the miRNAis endogenous to the cell or from a cell in which miRNA is exogenous tothe cell) and/or purified prior to labeling. This may achieve a reactionthat more efficiently labels the miRNA, as opposed to other RNA in asample in which the miRNA is not isolated or purified prior to labeling.In many embodiments of the invention, the label is non-radioactive.Generally, nucleic acids may be labeled by adding labeled nucleotides(one-step process) or adding nucleotides and labeling the addednucleotides (two-step process).

Moreover, miRNAs may be labeled as is described in U.S. PatentApplication Ser. No. 60/649,584, which is hereby incorporated byreference. Such nucleotides include those that can be labeled with adye, including a fluorescent dye, or with a molecule such as biotin.Labeled nucleotides are readily available; they can be acquiredcommercially or they can be synthesized by reactions known to those ofskill in the art.

Nucleotides for Labeling

Nucleotides for labelling are not naturally occurring nucleotides, butinstead, refer to prepared nucleotides that have a reactive moiety onthem. Specific reactive functionalities of interest include: amino,sulfhydryl, sulfoxyl, aminosulfhydryl, azido, epoxide, isothiocyanate,isocyanate, anhydride, monochlorotriazine, dichlorotriazine, mono- ordihalogen substituted pyridine, mono- or disubstituted diazine,maleimide, epoxide, aziridine, sulfonyl halide, acid halide, alkylhalide, aryl halide, alkylsulfonate, N-hydroxysuccinimide ester, imidoester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)-propionamide, glyoxal, aldehyde, iodoacetyl, cyanomethyl ester,p-nitrophenyl ester, o-nitrophenyl ester, hydroxypyridine ester,carbonyl imidazole, and the other such chemical groups. In someembodiments, the reactive functionality may be bonded directly to anucleotide, or it may be bonded to the nucleotide through a linkinggroup. The functional moiety and any linker cannot substantially impairthe ability of the nucleotide to be added to the miRNA or to be labeled.Representative linking groups include carbon containing linking groups,typically ranging from about 2 to 18, usually from about 2 to 8 carbonatoms, where the carbon containing linking groups may or may not includeone or more heteroatoms, e.g. S, O, N etc., and may or may not includeone or more sites of unsaturation. Of particular interest in manyembodiments are alkyl linking groups, typically lower alkyl linkinggroups of 1 to 16, usually 1 to 4 carbon atoms, where the linking groupsmay include one or more sites of unsaturation. The functionalizednucleotides (or primers) used in the above methods of functionalizedtarget generation may be fabricated using known protocols or purchasedfrom commercial vendors, e.g., Sigma, Roche, Ambion, and IDT. Functionalgroups may be prepared according to ways known to those of skill in theart, including the representative information found in U.S. Pat. Nos.4,404,289; 4,405,711; 4,337,063 and 5,268,486, and Br. Pat. No.1,529,202, which are all incorporated by reference.

Amine-modified nucleotides are used in several embodiments of theinvention. The amine-modified nucleotide is a nucleotide that has areactive amine group for attachment of the label. It is contemplatedthat any ribonucleotide (G, A, U, or C) or deoxyribonucleotide (G, A, T,or C) can be modified for labeling. Examples include, but are notlimited to, the following modified ribo- and deoxyribo-nucleotides:5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and8-[(6-amino)butyl]-amino-ATP; N⁶-(4-amino)butyl-ATP,N⁶-(6-amino)butyl-ATP, N⁴-[2,2-oxy-bis-(ethylamine)]-CTP;N⁶-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP;5-propargylamino-CTP, 5-propargylamino-UTP; 5-(3-aminoallyl)-dUTP;8-[(4-amino)butyl]-amino-dATP and 8-[(6-amino)butyl]-amino-dATP;N-(4-amino)butyl-dATP, N⁶-(6-amino)butyl-dATP,N⁴-[2,2-oxy-to-(ethylamine)]-dCTP; N⁶-(6-Amino)hexyl-dATP;8-[(6-Amino)hexyl]-amino-dATP; 5-propargylamino-dCTP, and5-propargylamino-dUTP. Such nucleotides can be prepared according tomethods known to those of skill in the art. Moreover, a person ofordinary skill in the art could prepare other nucleotide entities withthe same amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP, ATP,dCTP, dGTP, dTTP, or dUTP in place of a 5-(3-aminoallyl)-UTP.

Labeling Techniques

In some embodiments, nucleic acids are labeled by catalytically addingto the nucleic acid an already labeled nucleotide or nucleotides. One ormore labeled nucleotides can be added to miRNA molecules. See U.S. Pat.No. 6,723,509, which is hereby incorporated by reference.

In other embodiments, an unlabeled nucleotide or nucleotides iscatalytically added to an miRNA, and the unlabeled nucleotide ismodified with a chemical moiety that enables it to be subsequentlylabeled, in embodiments of the invention, the chemical moiety is areactive amine such that the nucleotide is an amine-modified nucleotide.Examples of amine-modified nucleotides are well known to those of skillin the art, many being commercially available such as from Ambion,Sigma, Jena Bioscience, and TriLink.

In contrast to labeling of cDNA during its synthesis, the issue forlabeling miRNAs is how to label the already existing molecule. To thisend, we may use an enzyme capable of using a di- or tri-phosphateribonucleotide or deoxyribonucleotide as a substrate for its addition toan miRNA, a small RNA molecule. Moreover, in specific embodiments, itinvolves using a modified di- or triphosphate ribonucleotide, which isadded to the 3′ end of an miRNA. The source of the enzyme is notlimiting. Examples of sources for the enzymes include yeast,gram-negative bacteria such as E. coli, lactococcus lactis, and sheeppox virus.

Enzymes capable of adding such nucleotides include, but are not limitedto, poly(A) polymerase, terminal transferase, and polynucleotidephosphorylase. In specific embodiments of the invention, ligase iscontemplated as NOT being the enzyme used to add the label, and instead,a non-ligase enzyme is employed.

Poly(A) polymerase has been cloned from a number of organisms fromplants to humans. It has been shown to catalyze the addition ofhomopolymer tracts to RNA (Martin et al, RNA, 4(2):226-30, 1998).

Terminal transferase catalyzes the addition of nucleotides to the 3′terminus of a nucleic acid.

Polynucleotide phosphorylase can polymerize nucleotide diphosphateswithout the need for a primer.

Labels and Tags

miRNAs or miRNA probes may be labeled with a positron emitting(including radioactive), enzymatic, colorimetric (includes visible andUV spectrum, including fluorescent), luminescent or other label or tagfor detection or isolation purposes. The label may be detected directlyor indirectly. Radioactive labels include ¹²⁵I, ³²P, ³³P, and ³⁵S.Examples of enzymatic labels include alkaline phosphatase, luciferase,horseradish peroxidase, and β-galactosidase. Labels can also be proteinswith luminescent properties, e.g., green fluorescent protein andphicoerythrin.

The colorimetric and fluorescent labels contemplated for use asconjugates include, but are not limited to, AMCA, Alexa Fluor dyes,BODIPY dyes, such as BODIPY FL, BODIPY 630/650, BODIPY 650/665, BODIPY-R6G, BODIPY-TRX; Cascade Blue; Cascade Yellow; coumarin and itsderivatives, such as 7-amino-4-methylcoumarin, aminocoumarin andhydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins anderythrosins; fluorescein and its derivatives, such as fluoresceinisothiocyanate; macrocyclic chelates of lanthanide ions, such as QuantumDye™; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red,tetramethylrhodamine and rhodamine 6G; Texas Red;

Specific examples of dyes include, but are not limited to, thoseidentified above and the following: Alexa Fluor 350, Alexa Fluor 405,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750;amine-reactive BODIPY dyes, such as BODIPY 493/503, BODEPY 530/550,BODEPY 558/568, BODIPY 564/570, BODDPY 576/589, BODIPY 581/591, BODEPY630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODEPY TMR, and,BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE,Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA,2′,4′,5′,7′-Tetrabromosulfonefluorescein, and TET.

Specific examples of fluorescently labeled ribonucleotides are availablefrom Molecular Probes, and these include, Alexa Fluor 488-5-UTP,Fluorescein-12-UTP, BODEPY FL-14-UTP, BODIPY TMR-14-UTP,Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, andBODIPY TR-14-UTP. Other fluorescent ribonucleotides are available fromAmersham Biosciences, such as Cy3-UTP and Cy5-UTP. Examples offluorescently labeled deoxyribonucleotides include Dinitrophenyl(DNP)-11-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP,Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODEPY FL-14-dUTP,Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODEPY TMR-14-dUTP,Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODEPY TR-14-dUTP,Alexa Fluor 594-5-dUTP, BODEPY 630/650-14-dUTP, BODIPY 650/665-14-dUTP;Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor594-7-OBEA-dCTP, Alexa Fluor 647-12-OBEA-dCTP. It is contemplated thatnucleic acids may be labeled with two different labels.

It is contemplated that synthetic miRNAs may be labeled with more thanone label, or with two different labels. Furthermore, fluorescenceresonance energy transfer (FRET) may be employed in methods of theinvention (e.g., Klostermeier et al., 2002; Emptage, 2001; Didenko,2001, each incorporated by reference). Fluorescent energy transfer dyes,such as thiazole orange-ethidium heterodimer; and, TOTAB may be used.Alternatively, the label may not be detectable per se, but indirectlydetectable or allowing for the isolation or separation of the targetednucleic acid. For example, the label could be biotin, digoxigenin,polyvalent cations, chelator groups and the other ligands, includeligands for an antibody.

Visualization Techniques

A number of techniques for visualizing or detecting labeled nucleicacids are readily available. The reference by Stanley T. Crooke, 2000has a discussion of such techniques (Chapter 6), which is incorporatedby reference. Such techniques include, microscopy, arrays, Fluorometry,Light cyclers or other real time PCR™ machines, FACS analysis,scintillation counters, Phosphoimagers, Geiger counters, MRI, CAT,antibody-based detection methods (Westerns, immunofluorescence,immunohistochemistry), histochemical techniques, HPLC (Griffey et al,1997, spectroscopy, capillary gel electrophoresis (Cummins et ah, 1996),spectroscopy; mass spectroscopy; radiological techniques; and massbalance techniques. Alternatively, nucleic acids may be labeled ortagged to allow for their efficient isolation. In other embodiments ofthe invention, nucleic acids are biotinylated.

When two or more differentially colored labels are employed, fluorescentresonance energy transfer (FRET) techniques may be employed tocharacterize the dsRNA. Furthermore, a person of ordinary skill in theart is well aware of ways of visualizing, identifying, andcharacterizing labeled nucleic acids, and accordingly, such protocolsmay be used as part of the invention. Examples of tools that may be usedalso include fluorescent microscopy, a BioAnalyzer, a plate reader,Storm (Molecular Dynamics), Array Scanner, FACS (fluorescent activatedcell sorter), or any instrument that has the ability to excite anddetect a fluorescent molecule.

Array Preparation

The present invention can be employed with miRNA arrays, which areordered macroarrays or microarrays of nucleic acid molecules (probes)that are fully or nearly complementary or identical to a plurality ofmiRNA molecules or precursor miRNA molecules and that are positioned ona support material in a spatially separated organization. Macroarraysare typically sheets of nitrocellulose or nylon upon which probes havebeen spotted. Microarrays position the nucleic acid probes more denselysuch that up to 10,000 nucleic acid molecules can be fit into a regiontypically 1 to 4 square centimeters. Microarrays can be fabricated byspotting nucleic acid molecules, e.g., genes, oligonucleotides, etc.,onto substrates or fabricating oligonucleotide sequences in situ on asubstrate. Spotted or fabricated nucleic acid molecules can be appliedin a high density matrix pattern of up to about 30 non-identical nucleicacid molecules per square centimeter or higher, e.g. up to about 100 oreven 1000 per square centimeter. Microarrays typically use coated glassas the solid support, in contrast to the nitrocellulose-based materialof filter arrays. By having an ordered array of miRNA-complementingnucleic acid samples, the position of each sample can be tracked andlinked to the original sample. A variety of different array devices inwhich a plurality of distinct nucleic acid probes are stably associatedwith the surface of a solid support are known to those of skill in theart. Useful substrates for arrays include nylon, glass and silicon Sucharrays may vary in a number of different ways, including average probelength, sequence or types of probes, nature of bond between the probeand the array surface, e.g. covalent or non-covalent, and the like.

Representative methods and apparatus for preparing a microarray havebeen described, for example, in U.S. Pat. Nos. 5,143,854; 5,202,231;5,242,974; 5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087;5,424,186; 5,429,807; 5,432,049; 5,436,327; 5,445,934; 5,468,613;5,470,710; 5,472,672; 806; 5,525,464; 5,503,980; 5,510,270; 5,525,464;5,527,681; 5,529,756; 5,532,128; 5,545,531; 5,547,839; 5,554,501;5,556,752; 5,561,071; 5,571,639; 5,580,726; 5,580,732; 5,593,839;5,599,695; 5,599,672; 5,610,287; 5,624,711; 5,631,134; 5,639,603;5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972; 5,695,940;5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645; 5,837,196;5,871,928; 5,847,219; 5,876,932; 5,919,626; 6,004,755; 6,087,102;6,368,799; 6,383,749; 6,617,112; 6,638,717; 6,720,138, as well as WO93/17126; WO 95/11995; WO 95/21265; WO 95/21944; WO 95/35505; WO96/31622; WO 97/10365; WO 97/27317; WO 99/35505; WO 09923256; WO09936760; WO0138580; WO 0168255; WO 03020898; WO 03040410; WO 03053586;WO 03087297; WO 03091426; WO03100012; WO 04020085; WO 04027093; EP 373203; EP 785 280; EP 799 897 and UK 8 803 000; the disclosures of whichare all herein incorporated by reference. It is contemplated that thearrays can be high density arrays, such that they contain 100 or moredifferent probes. It is contemplated that they may contain 1000, 16,000,65,000, 250,000 or 1,000,000 or more different probes. The probes can bedirected to targets in one or more different organisms. Theoligonucleotide probes range from 5 to 50, 5 to 45, 10 to 40, or 15 to40 nucleotides in length in some embodiments, hi certain embodiments,the oligonucleotide probes are 20 to 25 nucleotides in length.

The location and sequence of each different probe sequence in the arrayare generally known. Moreover, the large number of different probes canoccupy a relatively small area providing a high density array having aprobe density of generally greater than about 60, 100, 600, 1000, 5,000,10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes percm². The surface area of the array can be about or less than about 1,1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm².

Moreover, a person of ordinary skill in the art could readily analyzedata generated using an array. Such protocols are disclosed above, andinclude information found in WO 9743450; WO 03023058; WO 03022421; WO03029485; WO03067217; WO 03066906; WO 03076928; WO 03093810; WO03100448A1, all of which are specifically incorporated by reference.

Recently, alternative profiling methods have become available, based onsolution hybridization and subsequent immobilization and identificatione.g. Illumina platform.

Sample Preparation

It is contemplated that the miRNA of a wide variety of samples can beanalyzed using assays described herein. While endogenous miRNA iscontemplated for use with some embodiments, recombinant or syntheticmiRNA—including nucleic acids that are identical to endogenous miRNA orprecursor miRNA—can also be handled and analyzed as described herein.Samples may be biological samples, in which case, they can be fromblood, CSF, tissue, organs, tumor, semen, sputum, stool, urine, saliva,tears, other bodily fluid, hair follicles, skin, or any samplecontaining or constituting biological cells. Alternatively, the samplemay not be a biological sample, but be a chemical mixture, such as acell-free reaction mixture (which may contain one or more biologicalenzymes).

Cell Assays to Identify miRNAs with Ties to Disease

Specifically contemplated applications include identifying miRNAs thatcontribute to EMT that are themselves parts of a disease or conditionsor might otherwise be associated with a particular disease state.Additionally, a contemplated application includes the identification ofmiRNAs that are able to revert EMT and induce MET. Also, miRNA functionsmay be compared between a sample believed to be susceptible to aparticular disease or condition associated with EMT and one believed tobe not susceptible or resistant to that disease or condition. It isspecifically contemplated that RNA molecules of the present inventioncan be used to treat any of the diseases or conditions discussed in theprevious section or modulate any of the cellular pathways discussed inthe previous section. Specifically contemplated applications includeidentifying miRNAs that contribute to EMT cellular processes that arethemselves parts of a disease or might otherwise be associated with aparticular disease state. Also, miRNA functions may be compared betweena sample believed to be susceptible to a particular disease or conditionassociated with EMT and one believed to be not susceptible or resistantto that disease or condition.

The efficacy of different therapeutic drugs may be altered by miRNAs asdefined and used according to the present invention. Moreover, it hasbeen described that tumor cells that have undergone EMT may becomeresistant to chemo and immunotherapy (Thiery et al. 2009 Cell139:871-90). Therefore, miRNA based drugs that induce reversal of EMTmay enhance susceptibility to e.g. chemo and immunotherapy. Suchtherapeutic drugs include, but are not limited to, chemotherapeuticdrugs. A “chemotherapeutic agent” is used to connote a compound orcomposition that is administered in the treatment of cancer. Theseagents or drugs are categorized by their mode of activity within a cell,for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.Most chemotherapeutic agents fall into the following categories:alkylating agents, antimetabolites, antitumor antibiotics, mitoticinhibitors, and nitrosoureas.

Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma and calicheamicin omega);dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholmo-doxorubicm, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine;6-thioguanine; mercaptopurine; methotrexate; platinum coordinationcomplexes such as cisplatin, oxaliplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-Il);topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY1 17018, onapristone, and toremifene; aromatase inhibitorsthat inhibit the enzyme aromatase, which regulates estrogen productionin the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, megestrol acetate, exemestane, formestanie,fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens suchas flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; aswell as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);antisense oligonucleotides, particularly those which inhibit expressionof genes in signaling pathways implicated in aberrant cellproliferation, such as, for example, PKC-α, Raf and H-Ras; ribozymessuch as a VEGF expression inhibitor and a HER2 expression inhibitor;vaccines such as gene therapy vaccines and pharmaceutically acceptablesalts, acids or derivatives of any of the above. A list of U.S. FDAapproved oncology drags with their approved indications can be found onthe World Wide Web ataccessdata.fda.gov/scripts/cder/onctools/druglist.cfm. Moreover, it iscontemplated that samples that have differences in the activity ofcertain pathways may also be compared. Such cellular pathways includebut are not limited to the following: any adhesion or motility pathwayincluding but not limited to those involving cyclic AMP, protein kinaseA, G-protein couple receptors, adenylyl cyclase, L-selectin, E-selectin,PECAM, VCAM-I, α-actinin, paxillin, cadherins, AKT, integrin-α,integrin-β, RAF-I, ERK, PI-3 kinase, vinculin, matrixmetalloproteinases, Rho GTPases, p85, trefoil factors, profilin, FAK,MAP kinase, Ras, caveolin, calpain-1, calpain-2, epidermal growth factorreceptor, ICAM-1, ICAM-2, cofilin, actin, gelsolin, Rho A, Rac, myosinlight chain kinase, platelet-derived growth factor receptor or ezrin;any apoptosis pathway including but not limited to those involving AKT,Fas ligand, NFKB, caspase-9, PB kinase, caspase-3, caspase-7, ICAD, CAD,EndoG, Granzyme B, Bad, Bax, Bid, Bak, APAF-I, cytochrome C, p53, ATM,Bcl-2, PARP, Chk1, Chk2, Rho-21, c-Jun, Rho73, Rad51, Mdm2, Rad50,c-Abl, BRCA-I, perforin, caspase-4, caspase-8, caspase-6, caspase-1,caspase-2, caspase-10, Rho, Jun kinase, Jun kinase kinase, Rip2,lamin-A, lamin-B1, Lamin-B2, Fas receptor, H2O2, Granzyme A, NADPHoxidase, HMG2, CD4, CD28, CD3, TRADD, IKK, FADD, GADD45, DR3 deathreceptor, DR4/5 death receptor, FLIPs, APO-3, GRB2, SHC, ERK, MEK,RAF-1, cyclic AMP, protein kinase A, E2F, retinoblastoma protein,Smac/Diablo, ACH receptor, 14-3-3, FAK, SODD, TNF receptor, RTP,cyclin-D1, PCNA, BcI-XL, PIP2, PIP3, PTEN, ATM, Cdc2, protein kinase C,calcineurin, IKKα, IKKβ, IKKγ, SOS-I, c-FOS, Traf-1, Traf-2, WO or theproteasome; any cell activation pathway including but not limited tothose involving protein kinase A, nitric oxide, caveolin-1, actin,calcium, protein kinase C, Cdc2, cyclin B, Cdc25, GRB2, SRC proteinkinase, ADP-ribosylation factors (ARFs), phospholipase D, AKAP95, p68,Aurora B, CDK1, Eg7, histone H3, PKAc, CD80, PI3 kinase, WASP, Arp2,Arp3, p34, p20, PP2A, angiotensin, angiotensin-converting enzyme,protease-activated receptor-1, protease-activated receptor-4, Ras,RAF-I, PLCβ, PLCγ, COX-I, G-protein-coupled receptors, phospholipase A2,IP3, SUMO1, SUMO 2/3, ubiquitin, Ran, Ran-GAP, Ran-GEF, p53,glucocorticoids, glucocorticoid receptor, components of the SWI/SNFcomplex, RanBP1, RanBP2, importins, exportins, RCC1, CD40, CD40 ligand,p38, DCKα, IKKβ, NFKB, TRAF2, TRAF3, TRAFS, TRAF6, IL-4, IL-4 receptor,CDK5, AP-I transcription factor, CD45, CD4, T cell receptors, MAPkinase, nerve growth factor, nerve growth factor receptor, c-Jun, c-Fos,Jun kinase, GRB2, SOS-I, ERK-I, ERK, JAK2, STAT4, IL-12, IL-12 receptor,nitric oxide synthase, TYK2, IFNγ, elastase, IL-8, epithelins, IL-2,IL-2 receptor, CD28, SMAD3, SMAD4, TGFβ or TGFβ receptor; any cell cycleregulation, signaling or differentiation pathway including but notlimited to those involving TNFs, SRC protein kinase, Cdc2, cyclin B,Grb2, Sos-1, SHC, p68, Aurora kinases, protein kinase A, protein kinaseC, Eg7, p53, cyclins, cyclin-dependent kinases, neural growth factor,epidermal growth factor, retinoblastoma protein, ATF-2, ATM, ATR, AKT,CHK1, CHK2, 14-3-3, WEE1, CDC25 CDC6, Origin Recognition Complexproteins, p15, p16, p27, p21, ABL, c-ABL, SMADs, ubiquitin, SUMO, heatshock proteins, Wnt, GSK-3, angiotensin, p73 any PPAR, TGFα, TGFβ, p300,MDM2, GADD45, Notch, cdc34, BRCA-I, BRCA-2, SKP1, the proteasome, CUL1,E2F, pi 07, steroid hormones, steroid hormone receptors, IκBα, IκBβ,Sin3A, heat shock proteins, Ras, Rho, ERKs, IKIKs, PI3 kinase, Bcl-2,Bax, PCNA, MAP kinases, dynein, RhoA, PKAc, cyclin AMP, FAK, PIP2, PIP3,integrins, thrombopoietin, Fas, Fas ligand, PLK3, MEKs, JAKs, STATs,acetylcholine, paxillin calcineurin, p38, importins, exportins, Ran,Rad50, Rad51, DNA polymerase, RNA polymerase, Ran-GAP, Ran-GEF, NuMA,Tpx2, RCC1, Sonic Hedgehog, Crml, Patched (Ptc-1), MPF, CaM kinases,tubulin, actin, kinetochore-associated proteins, centromere-bindingproteins, telomerase, TERT, PP2A, c-MYC, insulin, T cell receptors, Bcell receptors, CBP, 1KB, NFKB, RAC1, RAF1, EPO, diacylglycerol, c-Jun,c-Fos, Jun kinase, hypoxia-inducible factors, GATA4, β-catenin,α-catenin, calcium, arrestin, survivin, caspases, procaspases, CREB,CREM, cadherins, PECAMs, corticosteroids, colony-stimulating factors,calpains, adenylyl cyclase, growth factors, nitric oxide, transmembranereceptors, retinoids, G-proteins, ion channels, transcriptionalactivators, transcriptional coactivators, transcriptional repressors,interleukins, vitamins, interferons, transcriptional corepressors, thenuclear pore, nitrogen, toxins, proteolysis, or phosphorylation; or anymetabolic pathway including but not limited to those involving thebiosynthesis of amino acids, oxidation of fatty acids, biosynthesis ofneurotransmitters and other cell signaling molecules, biosynthesis ofpolyamines, biosynthesis of lipids and sphingolipids, catabolism ofamino acids and nutrients, nucleotide synthesis, eicosanoids, electrontransport reactions, ER-associated degradation, glycolysis,fibrinolysis, formation of ketone bodies, formation of phagosomes,cholesterol metabolism, regulation of food intake, energy homeostasis,prothrombin activation, synthesis of lactose and other sugars,multi-drug resistance, biosynthesis of phosphatidylcholine, theproteasome, amyloid precursor protein, Rab GTPases, starch synthesis,glycosylation, synthesis of phoshoglycerides, vitamins, the citric acidcycle, IGF-I receptor, the urea cycle, vesicular transport, or salvagepathways. It is further contemplated that nucleic acids molecules of theinvention can be employed in diagnostic and therapeutic methods withrespect to any of the above pathways or factors. Thus, in someembodiments of the invention, a miRNA inhibits, eliminate, activates,induces, increases, or otherwise modulates one or more of the abovepathways or factors is contemplated as part of methods of the invention.The nucleic acid can be used to diagnosis a disease or condition basedon the relation of that miRNA to any of the pathways described above.

Other Assays

In addition to the use of arrays and microarrays, it is contemplatedthat a number of difference assays could be employed to analyze miRNAs,their activities and their effects. Such assays include, but are notlimited to, RT-PCR, in situ hybridization, hybridization protectionassay (HPA)(GenProbe), branched DNA (bDNA) assay (Collins, M. L. et al.(1997). Nucleic Acids Research 25: 2979-2984), rolling circleamplification (RCA), single molecule hybridization detection (USGenomics), Invader assay (ThirdWave Technologies), and Bridge LitigationAssay (Qiagen). It is contemplated that such methods may be used in thecontext of arrays, as well as in the context of diagnostic assays.

Therapeutic and Diagnostic Applications

miRNAs that affect phenotypic traits provide intervention points fortherapeutic applications as well as diagnostic applications (byscreening for the presence or absence of a particular miRNA). It isspecifically contemplated that RNA molecules of the present inventioncan be used to treat any of the diseases or conditions discussed in theprevious section. Moreover, any of the methods described above can alsobe employed with respect to therapeutic and diagnostic aspects of theinvention. For example, methods with respect to detecting miRNAs orscreening for them can also be employed in a diagnostic context. Intherapeutic applications, an effective amount of the miRNAs of thepresent invention is administered to a cell, which may or may not be inan animal. In some embodiments, a therapeutically effective amount ofthe miRNAs of the present invention is administered to an individual forthe treatment of disease or condition. The term “effective amount” asused herein is defined as the amount of the molecules of the presentinvention that are necessary to result in the desired physiologicalchange in the cell or tissue to which it is administered. The term“therapeutically effective amount” as used herein is defined as theamount of the molecules of the present invention that achieves a desiredeffect with respect to a disease or condition associated with EMT asearlier defined herein. A skilled artisan readily recognizes that inmany cases the molecules may not provide a cure but may provide apartial benefit, such as alleviation or improvement of at least onesymptom. In some embodiments, a physiological change having some benefitis also considered therapeutically beneficial. Thus, in someembodiments, an amount of molecules that provides a physiological changeis considered an “effective amount” or a “therapeutically effectiveamount.”

In some embodiments a molecule has a sequence that corresponds to themiRNA sequence from that particular animal, as opposed to from anotheranimal. Thus, in some embodiments, a human sequence is utilized as a RNAmolecule of the present invention. In in vivo experiments, a miRNAsequence used in a test animal may differ from a corresponding humansequence. In that case, a miRNA that differs from the human sequencemight be used to demonstrate therapeutic effect in the animal. Resultsobtained with this sequence tested in an animal may be extrapolatedexpected results in human with a corresponding miRNA molecule.

Modes of Administration and Formulations

The nucleic acid molecules of the invention may be administered to asubject alone or in the form of a pharmaceutical composition for thetreatment of a condition or disease. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the miRNA into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. For topical administration the miRNAs of theinvention may be formulated as solutions, gels, ointments, creams,suspensions, etc. as are well-known in the art. Systemic formulationsinclude those designed for administration by injection, e.g.subcutaneous, intravenous, intramuscular, intrathecal or intraperitonealinjection, as well as those designed for transdermal, transmucosal,inhalation, oral or pulmonary administration. For injection, the nucleicacids of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the nucleic acid molecules maybe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. For transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art. For oraladministration, the nucleic acids can be readily formulated by combiningthe molecules with pharmaceutically acceptable carriers well known inthe art. Such carriers enable the nucleic acids of the invention to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a patient tobe treated. For oral solid formulations such as, for example, powders,capsules and tablets, suitable excipients include fillers such assugars, e.g. lactose, sucrose, mannitol and sorbitol; cellulosepreparations such as maize starch, wheat starch, rice starch, potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques. For oral liquid preparationssuch as, for example, suspensions, elixirs and solutions, suitablecarriers, excipients or diluents include water, glycols, oils, alcohols,etc. Additionally, flavoring agents, preservatives, coloring agents andthe like may be added. For buccal administration, the molecules may takethe form of tablets, lozenges, etc. formulated in conventional manner.For administration by inhalation, the molecules for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the nucleic acids and a suitable powder basesuch as lactose or starch. The RNA molecules may also be formulated inrectal or vaginal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

In addition to the formulations described previously, the molecules mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, themolecules may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Alternatively, other pharmaceutical deliverysystems may be employed.

Liposomes and emulsions are well-known examples of delivery vehiclesthat may be used to deliver nucleic acids of the invention.

A nucleic acid of the invention may be administered in combination witha carrier or lipid to increase cellular uptake. For example, theoligonucleotide may be administered in combination with a cationiclipid. Examples of cationic lipids include, but are not limited to,lipofectin, DOTMA, DOPE, and DOTAP. The publication of WO0071096, whichis specifically incorporated by reference, describes differentformulations, such as a DOTAP; cholesterol or cholesterol derivativeformulation that can effectively be used for gene therapy. Otherdisclosures also discuss different lipid or liposomal formulationsincluding nanoparticles and methods of administration; these include,but are not limited to, U.S. Patent Publication 20030203865,20020150626, 20030032615, and 20040048787, which are specificallyincorporated by reference to the extent they disclose formulations andother related aspects of administration and delivery of nucleic acids.Methods used for forming particles are also disclosed in U.S. Pat. Nos.5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and5,972,900, which are incorporated by reference for those aspects. Thenucleic acids may also be administered in combination with a cationicamine such as poly-L-lysine. Nucleic acids may also be conjugated to achemical moiety, such as transferrin and cholesteryls. In addition,oligonucleotides may be targeted to certain organs or tissues by linkingspecific chemical groups to the oligonucleotide. For example, linkingthe oligonucleotide to a suitable array of mannose residues will targetthe oligonucleotide to the liver. Other targeting ligands are describedin Liu B., Brief Funct. Genomic Proteomic 6:112-119, 2007. Additionalexamples are carbohydrate sugars such as galactose,N-acetylgalactosamine, mannose; vitamins such as folates; smallmolecules including naproxen, ibuprofen or other known protein-bindingmolecules, cyclodextrin, which targets the transferrin receptor, alsocalled transferring modified cyclodextrin (Hu-Lieskovan et al., 2005),PEI (RGD-targeted PEG-PEI, Schiffelers et al. 2004), anisamide,RGD-peptide or RGD mimics, poly-arginin, anti-TfR single chain antibodyfragment/TfRscFv, Annexin A5 (targeting phophatidylserine exposingmembranes, Gamier B. et al., Bioconjug Chem., 2009, 11:2114-22), WO2009/126933 describing compositions and methods for site-specificdelivery of nucleic acids by combining them with targeting ligands andendosomolytic components. Targeting ligands that are preferentiallysuitable are tumor associated cell surface proteins, more preferablyprostate tumor associated cell surface proteins. Targeting of nucleicacids may also be accomplished by using aptamer technology as describedin WO2005/111238. Moreover, additional lipid moieties, such asPEG-lipids, cholesterol, endosomolytic helper lipids or peptides(WO2009/046220) or the overall morphology of the generated nanoparticles(characterized by charge and particle size) to the above mentioneddelivery vehicles may confer targeting specificity to either cancercells and/or tumor vasculature.

Additionally, the molecules may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the molecules for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the chimericmolecules, additional strategies for molecule stabilization may beemployed.

Alternatively, the molecules may be delivered using a coordinationchemistry based delivery system as described in WO2007011217, which isspecifically incorporated herein by reference.

Nucleic acids may be included in any of the above-described formulationsas the free acids or bases or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts are those salts that substantiallyretain the biological activity of the free bases and which are preparedby reaction with inorganic acids. Pharmaceutical salts tend to be moresoluble in aqueous and other protic solvents than are the correspondingfree base forms.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more miRNA molecules dissolved or dispersedin a pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce or produce acceptable adverse, allergicor other untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. Whether certain adverse effects areacceptable is determined based on the severity of the disease. Thepreparation of an pharmaceutical composition that contains at least onechimeric polypeptide or additional active ingredient will be known tothose of skill in the art in light of the present disclosure, asexemplified by Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference. Moreover, foranimal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The miRNAs may comprise different types of carriers depending on whetherit is to be administered in solid, liquid or aerosol form, and whetherit need to be sterile for such routes of administration as injection.The present invention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g. aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal or a patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, or 2% to 75% of the weight of the unit or 25% to 60% forexample and any range derivable therein. In other non-limiting examples,a dose may also comprise less than 1 microgram/kg/body weight, or 1microgram/kg/body weight, from 5 microgram/kg/body weight, 10microgram/kg/body weight, 50 microgram/kg/body weight, 100microgram/kg/body weight, 200 microgram/kg/body weight, 350microgram/kg/body weight, 500 microgram/kg/body weight, 1milligram/kg/body weight, 5 milligram/kg/body weight, 10milligram/kg/body weight, 50 milligram/kg/body weight, 100milligram/kg/body weight, 200 milligram/kg/body weight, 350milligram/kg/body weight, or 500 milligram/kg/body weight, to 1000mg/kg/body weight or more per administration, and any range derivabletherein. In non-limiting examples of a derivable range from the numberslisted herein, a range of 5 mg/kg/body weight to 100 mg/kg/body weight,5 microgram/kg/body weight to 500 milligram/kg/body weight, etc., can beadministered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The molecules may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines. In certain embodiments, the molecules areprepared for administration by such routes as oral ingestion. In theseembodiments, the solid composition may comprise, for example, solutions,suspensions, emulsions, tablets, pills, capsules (e.g., hard or softshelled gelatin capsules), sustained release formulations, buccalcompositions, troches, elixirs, suspensions, syrups, wafers, orcombinations thereof. Oral compositions may be incorporated directlywith the food of the diet. Preferred carriers for oral administrationcomprise inert diluents, assimilable edible carriers or combinationsthereof. In other aspects of the invention, the oral composition may beprepared as a syrup or elixir. A syrup or elixir, and may comprise, forexample, at least one active agent, a sweetening agent, a preservative,a flavoring agent, a dye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc. or combinations of the foregoing. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, carriers such as a liquid carrier. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

Any embodiment discussed above with respect to delivery or transport tocells can also be employed with respect to implementing delivery ofmedicinal compounds discussed in this section.

Effective Dosages

The molecules of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat or prevent adisease condition, the molecules of the invention, or pharmaceuticalcompositions thereof, are administered or applied in a therapeuticallyeffective amount. A therapeutically effective amount is an amounteffective to ameliorate or prevent the symptoms, or prolong the survivalof the patient being treated. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the EC50 as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the molecules which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from 0.01 to 0.1 mg/kg/day, or from 0.1 to 5 mg/kg/day,preferably from 0.5 to 1 mg/kg/day or more. Therapeutically effectiveserum levels may be achieved by administering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the proteins may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The amount of molecules administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs or treatment (including surgery).

Toxicity

Preferably, a therapeutically effective dose of the molecules describedherein will provide therapeutic benefit without causing substantialtoxicity. Toxicity of the molecules described herein can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD50 (the dose lethal to 50% of thepopulation) or the LD100 (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. Proteins which exhibit high therapeutic indices are preferred.The data obtained from these cell culture assays and animal studies canbe used in formulating a dosage range that is not toxic for use inhuman. The dosage of the proteins described herein lies preferablywithin a range of circulating concentrations that include the effectivedose with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl et al, 1975, In: The Pharmacological Basisof Therapeutics, Ch. 1, p. 1).

Pendant Groups

A “pendant group” may be attached or conjugated to the nucleic acid.Pendant groups may increase cellular uptake of the nucleic acid. Pendantgroups can be linked to any portion of the nucleic acid but are commonlylinked to the end(s) of the oligonucleotide chain. Examples of pendantgroups include, but are not limited to: acridine derivatives (i.e.2-methoxy-6-chloro-9-ammoacridine); cross-linkers such as psoralenderivatives, azidophenacyl, proflavin, and azidoproflavin; artificialendonucleases; metal complexes such as EDTA-Fe(II),o-phenanthroline-Cu(I), and porphyrin-Fe(II); alkylating moieties;nucleases such as amino-1-hexanolstaphylococcal nuclease and alkalinephosphatase; terminal transferases; abzymes; cholesteryl moieties;lipophilic carriers; peptide conjugates; long chain alcohols; phosphateesters; amino; mercapto groups; radioactive markers; nonradioactivemarkers such as dyes; and polylysine or other polyamines. In oneexample, the nucleic acid is conjugated to a carbohydrate, sulfatedcarbohydrate, or glycan.

Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, individual miRNAs are included in a kit. The kitmay further include one or more negative control synthetic miRNAs thatcan be used to control for the effects of synthetic miRNA delivery. Thekit may further include water and hybridization buffer to facilitatehybridization of the two strands of the synthetic miRNAs. The kit mayalso include one or more transfection reagent(s) to facilitate deliveryof the miRNA to cells.

In another non-limiting example, multiple synthetic miRNAs are includedin a kit. The kit may further include one or more negative controlsynthetic miRNAs that can be used to control for the effects ofsynthetic miRNA delivery. The kit may also include one or moretransfection reagents to facilitate delivery into cells.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit (labelingreagent and label may be packaged together), the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a vial. The kits of thepresent invention also will typically include a means for containing thenucleic acids, and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which thenucleic acid formulations are placed, preferably, suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent. Thekits of the present invention will also typically include a means forcontaining the vials in close confinement for commercial sale, such as,e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Such kits may also include components that preserve or maintain themiRNA or that protect against its degradation. Such components may beRNAse-free or protect against RNAses. Such kits generally will comprise,in suitable means, distinct containers for each individual reagent orsolution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented.

Kits of the invention may also include one or more of the following:miRNA, library of miRNAs, combination library of miRNA, negative controlmiRNA, nuclease-free water; RNase-free containers, such as 1.5 ml tubes;hybridization buffer; and transfection reagent(s).

It is contemplated that such reagents are embodiments of kits of theinvention. Such kits, however, are not limited to the particular itemsidentified above and may include any reagent used for the manipulationor characterization of miRNA.

Sequence Identity

“Sequence identity” is herein defined as a relationship between two ormore nucleic acid (nucleotide, polynucleotide, RNA, DNA) sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between nucleic acid sequences, asthe case may be, as determined by the match between strings of suchsequences.

In a preferred embodiment, identity also means identity percentage andcan be calculated by the number of equal nucleotides between subject andquery, divided by the total length of the query, and multiplied by 100.

“Identity” and “similarity” can be readily calculated by known methods,including but not limited to those described in Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heine, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the GCG program package (Devereux,J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP,BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410(1990). The BLAST X program is publicly available from NCBI and othersources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). Thewell-known Smith Waterman algorithm may also be used to determineidentity.

Preferred parameters for nucleic acid comparison include the following:Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970);Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap LengthPenalty: 3. Available as the Gap program from Genetics Computer Group,located in Madison, Wis. Given above are the default parameters fornucleic acid comparisons.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a miRNA molecule, an equivalentor a source thereof or a composition as defined herein may compriseadditional component(s) than the ones specifically identified, saidadditional component(s) not altering the unique characteristic of theinvention. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

DESCRIPTION OF THE FIGURES

FIGS. 1A-B: Flow chart of the screening protocol for EMT reversal, andoverview of the results obtained from one of the Lentivirus-basedmicroRNA expression library plates (ITM0081-0160). FIG. 1A, Lentivirusstock (1.0 ul; M01=3 to 300) was added to TSUpr1-pEcad-Luc cells for 24hours. Two days post infection (day 4), puromycin selection was applied,and six days after infection (day 8) luciferase activity was measured.FIG. 1B, The background-corrected firefly/Renilla luciferase ratios wereplotted against the lentiviral MOI. The four miRs with signalssignificantly above background (FLuc/RLuc ratio>average+2×standarddeviation) are encircled. The four control values represent FLuc/RLucratios induced by miR-141 and miR-200c, which were supplied in separatetubes (both in duplicate).

FIG. 2: Overview of the pEcad-Luc/Riuc expression vector. The CDH1 genepromoter (GenBank L34545, position −294 to +44), containing the threeE-boxes, was obtained by PCR amplification using human genomic DNA as atemplate. The CDH1 promoter fragment was cloned upstream of the fireflyluciferase gene in the pGL2-basic vector (Promega). The HSV-Tkpromoter-driven Renilla luciferase cassette was obtained from the pRL-TKvector (Promega), and the SV40 promoter-driven Zeocinantibiotics-resistance cassette from the pVgRXR vector (Invitrogen). TheRenilla luciferase and the ZeocinR cassettes were cloned into thepGL2-basic vector at the indicated locations.

FIG. 3: Characterization of tumor cell line models. Total RNA wasisolated from 70-80% confluent cell line cultures, and analyzed for CDH1and Vimentin expression by qPCR. Relative expression values wereplotted, and cells with high CDH1/low VIM and low CDH1/high VIM weredesignated epithelial and mesenchymal, respectively.

FIGS. 4A-C: MET induction using synthetic miRNA mimics. FIG. 4A,TSUpr1-pEcad-Luc cells were transiently transfected with 20 to 60 nM ofsynthetic miR mimics (Ambion or Dharmacon; see Table 7 for details).Four days post-transfection, Luc and RLuc activity were measured, andthe Luc/RLuc ratios were normalized for negative control(NC)-transfected cells. FIG. 4B, Cells were transfected and treated asdescribed in FIG. 4A. Total RNA was isolated and used for miRNA and geneqPCR analysis. Expression levels of CDH1 and CDH2, normalized to theHPRT housekeeping gene, as percentage of NC-transfected cells are shown.FIG. 4C, Different parental tumor cell lines were transfected withmiR-200c and miR-520f mimics and other miR mimics and analyzed for CDH1expression (as described in FIG. 4B).

FIGS. 5A-C: Creation of doxycycline inducible miRNA expression systems.FIG. 5A, The miR-200c and miR-520f precursors were isolated from theprovirus integrated into pCDH-lentivirus infected TSUpr1 cells (in themiR screening experiments) by restriction enzyme digestion. The miRprecursors were cloned into the NheI/EagI sites of the pmRi-ZsGreen1inducible miR expression vector (Clontech PT5049-1). Map of the miR-520fexpression vector is shown. FIG. 5B, PC3 cells (ATCC# CRL-1435) weretransfected with the pTet-on-advanced vector (Clontech PT3899-5), andselected in G418 (300 ug/ml)-containing medium. Cells were transientlytransfected with the pTRE-Luc reporter vector, treated with 0, 0.1 and1.0 ug/ml doxycycline (DOX, Sigma D9891) for 2 days, and then analyzedfor Luc activation. FIG. 5C, PC3-Tet-on cells (clone 8) were thentransfected with the pmRi-ZsGreen1-miR-200c or miR-520f vector, andselected in puromycin (5 ug/ml)-containing medium. Stable clones ofPC3-mRi-ZsGreen-miR-X were tested for the induction of miRNA and geneexpression. Cells were treated with 0 and 1.0 ug/ml DOX for 4 days,total RNA was isolated, and miRNA and gene expression (Table 8) wereanalyzed by qPCR.

FIGS. 6A-D Influence of miRNAs on tumor cell invasion. FIG. 6A, Fiftythousand (PC3) or fourty thousand (TSUpr1) cells were seeded intoBiocoat Matrigel Invasion chambers, 8 micron (BD 354480), in serum-freemedium. The invasion chamber was placed in a 24-well containing mediumwith 10% fetal calf serum as chemoattractant. As a control, the sameamount of cells was seeded onto the surface of a 24-well culture plate.After 48 hours incubation, cells in the invasion chamber were removed byaspiration and cleaning the inner compartment with a cotton swab. Theinvasion chamber was then put into CellTiter-GLO (CTG, Promega-G7571)cell viability reagent, and incubated for 15 minutes. CTG activity wasmeasured on a Victor3 luminometer. FIG. 6B, Cell invasion assays wereperformed with PC3-mRi-ZsGreen1-miR-X cells that were pre-treated for 2days with 1 ug/ml DOX. In addition, PC3 cells infected with miR-200c andmiR-520f lentivirus (MOI=30), and selected on puromycin for 4 days, wereused. The percentage of cell invasion was calculated as the CTG activityon the lower part of the membrane divided by the total CTG activity (ofthe cells grown on the surface of a 24 well culture plate). Inhibitionof cell invasion by a specific miRNA was calculated by dividing thepercentage of cell invasion versus untreated (−DOX) or control (emptyvector virus) cells. FIG. 6C, Relative microRNA expression levels inparallel cultures were determined by stem-loop RT-qPCR. FIG. 6D, Cellinvasion assays were performed with TSUpr-pEcad cells that were infectedwith empty-vector, miR-124-1, miR-181a-1, miR-200c, miR-206, miR-518b,miR-520f or miR-524 precursor-containing lentivirus (M01=30), andselected on puromycin for 4 days. Cell invasion was calculated asdescribed in FIG. 6B.

EXAMPLES Example 1 Material and Methods

Generation of the Lentiviral Library Encoding miRNAs

Human miRNAs were selected from both the public miRNA repository(www.mirbase.org) and proprietary small RNA deep sequencing data (see WO2007/081204). The miRNA sequences were amplified from their genomiclocation with amplicons containing the full-length pre-miRNA hairpin anda flanking sequence on both sides of 50-150 basepairs. The primers forthe amplicons were designed using Primer3 software (www.geneious.com).If the primer design program could not find appropriate primers in thedesignated sequences, the requirements for the flanking sequences wereadjusted to 0-200 basepairs. The designed primers were complemented witha 5′ GCGC overhang and a restriction site for directional cloning. Asdefault the primer upstream of the miRNA was complemented with a BamHIrestriction site (GGATCC) and the primer downstream of the miRNA wascomplemented with an EcoRI restriction site (GAATTC). Primers ofamplicons with internal BamHI or EcoRI restriction sites (i.e. occurringin the genomic sequence) were complemented with either a BglII site(AGATCT) or a XbaI site (TCTAGA) respectively. The miRNAs were amplifiedusing the abovementioned primers from human genomic DNA of a singleindividual in the following PCR reaction:

constituent concentration volume supplier/cat # buffer 10X   1 uLStratagene/600159 dNTPs  10 mM each 0.2 uL GE Healthcare/27-18(5-8)0-04fwd primer  10 uM 0.2 uL IDT (Integrated DNA Technologies) rev primer 10 uM 0.2 uL IDT (Integrated DNA Technologies) gDNA 100 ng/uL 0.1 uLprivate source Pfu DNA pol  2.5 U/uL 0.1 uL Stratagene/600159 H₂O N/A8.2 uL N/A temp (° C.) time cycles 95  2 min 95 15 s 40  59* 15 s 40 *−0.1° C./cycle 72 90 s 40 72 15 min  4 ∞

All miRNA loci were amplified in separate 10 uL PCR reactions. Theproducts were purified using the Qiagen PCR Clean-Up buffer set andWhatman Unifilter GF/C filter plates (cat #7700-1101). DNA was elutedwith 17 uL H₂O per well. The separate eluates were used in the followingrestriction reaction:

constituent concentration volume supplier/cat # buffer E 10X   2 uLPromega/R005A EcoRI* 12 U/uL 0.1 uL Promega/R6017 BamHI* 10 U/uL 0.1 uLPromega/R6025 eluate N/A  16 uL N/A H₂O N/A 1.8 uL N/A *Amplicons withinternal restriction sites for EcoRI or BamHI were cut with XbaI orBglII respectively instead. The EcoRI + BglII reaction was done withPromega buffer D. The BamHI + XbaI reaction was done with Promega bufferE.

Restriction for 2 hours at 37° C. The separate 20 uL restrictionreactions were purified using the Qiagen PCR Clean-Up buffer set andWhatman Unifilter GF/C filter plates (cat #7700-1101). DNA was elutedwith 20 uL H₂O per well. The separate eluates were used in the followingligation reaction:

constituent concentration volume supplier/cat # buffer 10X   2 uLPromega/C1263 T4 DNA ligase 1-3 U/uL 0.2 uL Promega/M1804 restrictedpCDH*   1 ng/uL 7.8 uL System Biosciences/ CD510B-1 eluate N/A  10 uLN/A Ligation overnight at 4° C. *For directional cloning, pCDH was cutwith both EcoRI and BamHI. An alternate construct called pCDH− was madewith reversed EcoRI and BamHI restriction sites so that the ampliconswith 5′ BamHI and 3′ EcoRI were cloned in the proper direction.

The amplicons with an internal EcoRI site were cut with XbaI and ligatedinto a pCDH vector that was restricted with XbaI and BamHI.

The resulting ligates were transformed separately into bacteria (PromegaSingle Step (KRX) competent cells, cat # L3002). 50 uL competent cellswas diluted with 950 uL transformation buffer II (10 mM MOPS, 75 mMCaCl₂, 10 mM RbCl, 15% glycerol, filter-sterilized). Per 20 uL ligate 20uL diluted competent cells was added. The mix was incubated for 15minutes on ice, heat-shocked at 37° C. for 30 seconds, and put back onice. After 2 minutes the transformed bacteria were reconstituted in 150uL lysogeny broth (LB). The bacteria were allowed to recover for 20minutes at 37° C. after which they were plated out separately onampicillin-containing (50 ug/mL) LB-agar plates and grown overnight at37° C.

Single colonies of each plate are picked and subcultured overnight in400 uL ampicillin-containinig (50 ug/mL) LB. 1 uL of subculture is lysedin 100 uL water for sequencing purposes. Bacterial lysate is used in thefollowing PCR reaction:

constit- concen- uent tration volume supplier/cat # buffer 5X 1 uLprivate source dNTPs 10 mM 0.1 uL GE Healthcare/ each 27-18(5-8) 0-04pCDH-fwd 10 uM 0.1 uL IDT (Inte- grated DNA Technologies) pCDH-rev 10 uM0.1 uL “ “ lysate 1:100 1 uL N/A Taq DNA pol un- 0.02 uL private sourceknown H₂O N/A 2.68 uL N/A temp (° C.) time cycles 95  2 min 95 15 s 4059* 15 s 40 *-0.1° C./cycle 72 90 s 40 72 15 min  4 ∞pCDH-fwd CACGCTGTTTTGACCTCCATAGA pCDH-rev CACTGACGGGCACCGGAG(SEQ ID NO: 84-85)

The PCR products were diluted 25×. 1 uL of diluted PCR product was usedin the following Sanger Sequencing reaction:

constit- concen- uent tration volume supplier/cat # buffer N/A 1.9 uLprivate source BigDye 10 mM 0.1 uL ABI/4336921 V3.1 pCDH-seq 10 uM0.1 uL IDT (Integrated DNA Technologies) PCR prod- 1:25 1 uL N/A uct H₂ON/A 1.9 uL N/A temp (° C.) time cycles 94 10 sec 50  5 s 40 60  2 min 4010 ∞ pCDH-seq GACCTCCATAGAAGATTCTAGAGCTAGC (SEQ ID NO: 86)

30 u precipitation mix (80% ethanol, 50 mM sodium acetate pH 5.5) wasadded to each of the sequencing reaction products. The mixes werevortexed for 10 seconds and spun down at 5000 ref for 45 minutes at 4°C. Supernatant was aspirated and DNA pellets were washed with 30 uL icecold 80% ethanol and spun at 5000 ref for 5 minutes at 4° C. Supernatantwas aspirated and the DNA pellet was dried on a heat block for 10minutes. The dry DNA pellet was dissolved in 10 uL H₂O. The resultingDNA solution was sequenced on an ABI 3730XL DNA Analyzer. Sequences werecompared to the expected genomic sequences. Correct clones were added tothe library. For incorrect clones an additional 4 bacterial colonieswere picked, and analyzed for insert sequence. Library constructs weresubcultured overnight in 50 mL ampicillin-containing (100 ug/mL) LB andisolated with the Qiagen QIAfilter Plasmid Midi Kit (cat #12245)supplemented with the Qiagen EndoFree Plasmid Buffer Set (cat #19048)according to the instructions of the manufacturer. DNA was dissolved inthe supplied TE buffer and brought to a final concentration of 500ng/uL.

We ordered constructs that we were not able to clone ourselves asminigenes from Integrated DNA Technologies. In these cases, thefull-length hairpin plus 20 basepairs flanking each site were clonedinto our vector as a service by IDT.

Packaging and virus production was performed by System Biosciences asdescribed in the user manual of CD-500B1-CD523-A1.

Cell Culture

The TSUpr1/pEcad-luc/Rluc cell line was generated by stable transfectionof the human transitional cell bladder carcinoma cell line TSUpr1 withthe pEcad-Luc/Rluc expression vector (FIG. 2). A single,zeocin-resistant, clone (clone 1.c.4) was used for all experiments.

TSUpr1/pEcad-luc/Rluc cells were maintained in RPMI-1640 medium(Invitrogen, 31870), supplemented with 10% Fetal Bovine Serum (Sigma,F7524), L-Glutamine (Invitrogen 25030-024) and 50 μg/ml Zeocin(Invitrogen, R250-01). Cells were maintained in a humidified atmosphereat 37° C. and 5% CO₂. Cells were split once a week at a 1:20 ratio.

Chemicals

Polybrene (2 μg/ml; Sigma, H9268) was used to increase the efficiency ofinfection with the miRNA-encoding lentiviral particles. Puromycin (5μg/ml; Sigma, P8833) was used to select for cells expressing the miRNAof interest. Zeocin (50 μg/ml; Invitrogen, R250-01) was used to maintainintegration and expression of the Ecad-luc/Rluc transgene.

MET Screening Protocol: Day 1: Cell Seeding

TSUpr1/pEcad-luc/Rluc cells were seeded at a density of 2,500 cells perwell in 96-well plates (100 μl total volume per well), in duplicate.

Day 2: Lentiviral Infection

The packaged lentiviral constructs were provided as frozen VSV-Gpseudotyped viral particles, and are stored at −80° C. Before use,contents were thawed at room temperature and put on ice immediatelyafterwards. To open tubes a SepraSeal Cap Removal Tool (FisherScientific Cat#: 50823908) was used and precipitates were resuspended bypipetting a few times. Medium was removed using a multichannel pipette.Gently, 200 μl of fresh medium (+FBS/glutamine/zeocin) containing 2μg/ml polybrene (Sigma, H9268) was added to the cells. Next, 1.0 μl ofundiluted lentiviral particles was added to each well (in duplicate). Oneach 96-well plate, miR-141 and miR-200c encoding lentiviral particleswere added as positive controls.

Day 3: Refresh Medium

Twenty-four hours after the addition of lentivirus, medium was removedusing a multichannel pipette. Cells were washed once with 0.9% NaCl, 50μl per well. Subsequently, 100 μl fresh medium (+FBS/glutamine/zeocin)was added to each well.

Day 4: Puromycin-Selection

Medium was removed using a multichannel pipette. To select fortransduced cells, 200 μl fresh medium containing 5 μg/ml Puromycin(Sigma, P8833) was added to the cells. Note, the cells were not washedin between.

Day 8: Dual-Luciferase Reporter Assay (Promega)

Medium was removed using a multichannel pipette. Next, cells were washedgently with 0.9% NaCl (50 μl/well). To prepare cell lysates, PassiveLysis Buffer 1× (Promega, E1980) was added to the cells (20 μl perwell), and incubated at room temperature for 20 minutes on a plateshaker. Cell lysates were transferred to a white 96-well microtiterplates (Thermo Scientific, 9502887). Firefly and Renilla luciferaseactivity were measured on a Victor³ Multilabel Counter (PerkinElmer),according to the manufacturer's instructions.

Total RNA Isolation

TSUpr1/pEcad-luc/Rluc cells were seeded at a density of 15,000 cells perwell in 24-well plates. Lentiviral infection was performed as describedabove. Due to the limited amount of viral particles, virus was added ata multiplicity of infection (MOI) of 30, and if possible an MOI of 100.At day 8, total RNA was isolated using Trizol reagent (200 μl per well),according to the manufacturer's instructions (Invitrogen, 15596-018).Concentration and purity of the RNA was determined on a Nanodrop-1000spectrophotometer (Thermo Scientific).

Real Time RT-PCR

Two micrograms of total RNA was DNase-I-treated (Invitrogen, 18068-015)and cDNA was synthesized using random hexamer primers and SuperScriptII-MMLV reverse transcriptase (Invitrogen, 18064-014). The RT-reaction(30 μl) was diluted 4 times in H₂O.

Gene expression was determined by SYBR Green qPCR, using SYBR Green PCRmix (Roche, 04707516001) and 2 μl cDNA as a template. RNA not subjectedto reverse transcriptase was used as a negative control for PCRamplification. Gene-specific primers are as follows:

E-Cadherin forward1 5′-GAAAAGAGAGTGGAAGTG-3′ reverse15′-GTGAAGGGAGATGTATTG-3′ E-cadherin forward2 5′-CAGGTCTCCTCTTGGCTCTG-3′reverse2 5′-ACTTTGAATCGGGTGTCGAG-3′ N-Cadherin forward5′-GAGGATTAGCCGGAACAACA-3′ reverse 5′-AACAAATTTCCCCCATCTCC-3′ SNAILforward 5′-AGGATCTCCAGGCTCGAAAG-3′ Reverse 5′-GACATCTGAGTGGGTCTGGA-3′SLUG forward 5′-TTCGGACCCACACATTACCT-3′ Reverse5′-TTGGAGCAGTTTTTGCACTG-3′ ZEB1 forward 5′-ATGCGGAAGACAGAAAATGG-3′reverse 5′-GTCACGTTCTTCCGCTTCTC-3′ ZEB2 forward5′-CGCTTGACATCACTGAAGGA-3′ reverse 5′-CTTGCCACACTCTGTGCATT-3′ β2Mforward 5′-AGCAGAGAATGGAAAGTCAAA-3′ reverse 5′-TGCTGCTTACATGTCTCG-3′(SEQ ID NO: 48-63).

Q-PCR was performed on a LightCycler LC480 instrument (Roche), using thefollowing amplification conditions: 5 min. 95° C., followed by 45 cyclesof 10 sec. 95° C., 20 sec. 60° C., 20 sec. 72° C. For E-cadherin primerset 1, an annealing temperature of 49° C. (instead of 60° C.) was used.Cp values were determined using the LightCycler 480 SW 1.5 software(Roche). Beta-2-microglobulin expression was used for normalization.Relative gene expression levels were calculated according to the modeldescribed by Pfaffl (18).

Stem-Loop RT-PCR

MicroRNA expression was determined by stem-loop RT-PCR as described(19). For this, 100 ng total RNA was reverse transcribed using 0.375pmol miR-specific stem-loop (SL) primers:

miR-141: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CCATCT-3′miR-200c: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC TCCATC-3′miR-181a-1: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACA CTCAC-3′miR-124*: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC GGCATT-3′miR-518b: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACA CCTCT-3′miR-520f: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC AACCCT-3′miR-524-5p: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC GAGAAA-3′miR-524-3p: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC ACTCCA-3′(SEQ ID NO: 64-71)in 1×RT buffer, containing 0.25 mM dNTPs, 3.33 U/μl SuperScript II-MMLVreverse transcriptase (Invitrogen) and 0.25 U/μl HRP-I RNase Inhibitor(Amersham), for 30 min. at 16° C., 30 min at 42° C. and 5 min at 85° C.Stem-loop RT products were diluted 2 times in H₂O.

MicroRNA expression was determined by SYBR Green qPCR, using PCR mix(0.5 μl forward primer (25 pmol/μl), 0.5 μl reverse primer (25 pmol/μl),8 μl H₂O, 10 μl 2× SYBR Green PCR mix, Roche) and 2 μl of stem-loop RTproduct as a template. RNA not subjected to SL-RT was used as a negativecontrol for PCR amplification. The miR-specific primers are as follows:

Forward Primers:

miR-141: 5′-GCCCGCTAACACTGTCTGGTAAAG-3′ miR-200c:5′-GCCCGCTAATACTGCCGGGTAATG-3′ miR-181a-1: 5′-TGCCAGAACATTCAACGCTGTCG-3′miR-124*: 5′-TGCCAGTAAGGCACGCGGTGA-3′ miR-518b:5′-TGCCAGCAAAGCGCTCCCCTTTAG-3′ miR-520f: 5′-GCCCGCAAGTGCTTCCTTTTAGAG-3′miR-524-5p: 5′-GCCCGCCTACAAAGGGAAGCACT-3′ miR-524-3p:5′-TGCCAGGAAGGCGCTTCCCTTTG-3′

A universal reverse primer was used: 5′-GTGCAGGGTCCGAGGT-3′ (SEQ ID NO:72-80)

Q-PCR was performed on a LightCycler LC480 instrument (Roche), using thefollowing amplification conditions: 5 min. 95° C., followed by 45 cyclesof 10 sec. 95° C., 20 sec. 60° C., 10 sec. 72° C. Cp values weredetermined using the LightCycler 480 SW 1.5 software (Roche). Expressionof the U6 snRNA (RNA6-1) was used for normalization (U6 (RNU6-1)primers: RT 5′-GTCATCCTTGCGCAGG-3′ U6 forward 5′-CGCTTCGGCAGCACATATAC-3′and U6 reverse 5′-AGGGGCCATGCTAATCTTCT-3′ (SEQ ID NO: 81-83). RelativemiR expression levels were calculated according to the model describedby Pfaffl (18).

DNA Sequence Analysis

The sequence of the cloned miRNAs in the lentiviral vectors for the hitsas described in Table 1 was verified as follows. Total nucleic acids oflentiviral transduced cells were isolated using Trizol reagent,according to the manufacturer's instructions (Invitrogen, 15596-018).Concentration and purity of the nucleic acids was determined on aNanodrop-1000 spectrophotometer (Thermo Scientific). Proviral DNA wasamplified by PCR, using 500 ng of nucleic acids as input, and pCDHlentiviral vector-specific primers (forward:5′-CACGCTGTTTTGACCTCCATAGA-3′, reverse: 5′-CACTGACGGGCACCGGAG-3′, (SEQID NO: 84-85).) for 35 cycles at an annealing temperature of 65° C.Amplified products were purified using the Wizard PCR preps DNApurification system (Promega). DNA sequence analysis was performed using2 of purified amplicon, 5 pmoles of pCDH-specific primer(5′-GACCTCCATAGAAGATTCTAGAGCTAGC-3′, (SEQ ID NO: 86).), and the Big DyeTerminator v 1.1 kit (Applied Biosystems). Products were analyzed on a3730 DNA Analyzers (Applied Biosystems). Data were collected using theCollection Software v3.0 and analyzed using the Sequencing Analysisv5.3.1 program (Applied Biosystems). The sequence for all cloned miRNAswas correct and is given in Table 4.

Results

Mesenchymal to Epithelial Transition (MET) microRNA Screening

EMT in tumor cells results from a transcriptional reprogramming of thecell. In particular the transcriptional repression of the E-cadherin(CDH1) gene promoter has been shown to trigger the EMT phenotype. TheE-cadherin protein is one of the most important cadherin moleculesmediating cell-cell contacts in epithelial cells/tissues. CDH1 isrepressed by binding of the transcriptional repressors, SNAI1, SNAI2,TCF3, TWIST, ZEB1 or ZEB2 (20-23), to three so-called E-boxes in theCDH1 proximal promoter region (24-26). Inhibiting the binding of theserepressors to the CDH1 promoter can revert EMT, also calledmesenchymal-to-epithelial transition (MET), and inhibits tumor cellinvasion and tumor progression in animal models (27). We hypothesizedthat an extensive set of microRNAs is able to induce MET, by targetingone of the EMT-associated transcriptional repressors. Suppression of theEMT-associated transcriptional repressors will result in there-activation of CDH1 gene expression. This re-activation of CDH1 can beeasily monitored by CDH1 promoter-drivenfirefly luciferase activation.Therefore, we cloned the core element of the CDH1 gene promoter,containing the three E-boxes (24; 25), in an expression vector to drivefirefly luciferase expression. As an internal control, a HSV-Tkpromoter-driven Renilla luciferase cassette was inserted into the samevector (pEcad-Luc). We have stably transfected the bladder cancer cellline TSUpr1 with this reporter construct. Endogenous CDH1 mRNAexpression in this cell line is undetectable, whilst several mesenchymalmarkers are expressed (24; 28).

microRNA-Induced MET Screening: SETUP

TSUpr1-pEcad-Luc cells are susceptible to puromycin selection. As afirst step in setting up the screening model, we determined thetransduction efficiency of TSU cells. TSU cells were infected with aeGFP-expressing lentivirus at different MOIs, and transduced cells wereselected in puromycin-containing medium. The number of infected, andhence puromycin-resistant, cells was determined by a MTT cell survivalassay. In two independent experiments, we have shown that at an MOI of8, a transduction efficiency (MTT of infected cell+puro/MTT of infectedcell-puro×100%) of at least 60% could be achieved. Based on theseresults, we decided to perform the pilot infection experiments at an MOIof 3 and 30.

Recently, the miR-200 family and miR-205 were shown to regulate EMT bytargeting ZEB1 and ZEB2 (13-15). Two miRNAs of the miR-200 family,miR-141 and miR-200c, were selected to setup and optimize our METscreening assay. TSU cells were infected with both miRNA vectors(M01=30). Two (day 4) and six (day 8) days post infection, E-cadherinpromoter-driven firefly luciferase activity was measured and normalizedagainst the HSV-Tk controlled Renilla luciferase activity. At day 8, theFLuc/RLuc ratio was induced more than 2-fold by miR-200c, and more than1.5-fold by miR-141 (FIG. 1; Table 1). To decrease the ‘noise’ ofuninfected cells (FLuc- and RLuc+), puromycin selection was applied. ThemiR-141 and miR-200c-induced FLuc/RLuc ratios were comparable to thosewithout selection.

TSU cells were infected with 0.2 microliter of undiluted miR-lentivirus.Two days after infection, puromycin selection was applied for 4 days.Six days post-infection luciferase activities were measured. The resultof screening of the first 80 microRNAs is shown in FIG. 1. The fourpositive ‘hits’ (FLuc/RLuc>average+2×SD) were microRNAs known toregulate EMT, i.e. miR-141, miR-200c, miR-205, and miR-429. This pilotexperiment indicated the validity of the screening system.

microRNA-Induced MET Screening

Several lentiviral constructs in the miR library have very high titers.Therefore, without dilution of virus stocks prior to infection, in somecases high MOIs of virus were applied. The addition of miR-141 andmiR-200c lentivirus at high MOI (100 to 600) had no significanttoxicity, whilst the induction of the FLuc/RLuc ratio was slightlyenhanced. Therefore, the entire lentivirus-based microRNA expressionlibrary was screened using one microliter of undiluted lentivirus (allin duplicate). After screening all 1120 miRs (14 plates, each containing80 miR vectors), 65 positive ‘hits’ (FLuc/RLuc>average+2×SD) were found.In addition to these 65 miRs, 59 additional miRs were selected based on,e.g. increased Luc signal with increased RLuc signal, leaving the ratiobelow the threshold. These 124 miRs were re-screened in theTSUpr1-pEcad-luc model, after which 30 miRs with a reproducible positiveFLuc/RLuc ratio remained (26 of 30 from the group of 65 ‘hits’).

Preliminary MET microRNA Validation

To further validate and select miRs relevant for EMT/MET regulation, westudied their effects on endogenous gene expression, i.e. CDH1(E-cadherin), CDH2 (N-cadherin), SNAI1 (SNAIL), SNAI2 (SLUG), ZEB1(deltaEF1) and ZEB2 (SIP1). TSUpr1-pEcad-luc cells were infected withthe 30 miRs identified by MET screening (MOI=30 and 100). Six days afterinfection, total RNA was isolated and used for qPCR analysis of theabove-mentioned genes (Table 1). The expected upregulation of CDH1(E-cadherin) expression was only observed with a few miRs (n=10), as wasthe downregulation of CDH2 (N-cadherin) expression (n=8). Of those miRs,miR-181a-1, miR-200a, mir-429 and miR-524 resulted in both upregulationof CDH1 and downregulation of CDH2. The kinetics of cadherin expressionby EMT-associated miRs is cadherin-dependent. This may explain thenon-consistent up and down regulation of both cadherins, at the chosentime point (6 days post miR introduction). The hallmark of EMT or thereverse, MET, is cadherin switching. Therefore, the (relative) ratio ofCDH1/CDH2 expression was used as a measure to study the role of theidentified miRs on EMT regulation. As shown in Table 1, ten miRs (out of12) had an increased CDH1/CDH2 ratio (range: 1.85-17.65). The other twomiRs induced CDH1 expression significantly (in line with the FLucinduction), but these miRs also substantially (>2-fold) induced CDH2expression. Of the 12 miRs that induced endogenous CDH1 expression orinduced a cadherin switch, all also down regulated at least one CDH1transcriptional repressor at the RNA level.

Conclusions

In the set of 12 selected miRs, the known EMT-associated miRs (miR-200family and miR-205) are present, which confirms the specificity of oursystem. Of the remaining 9 miRs, three miRs (miR-518b, miR-520f andmiR-524) belong to the human miR-515 family. In this study, the miR-200family members in general down regulated ZEB2 expression, which wasassociated with down regulation of CDH2 expression (at day 8). On theother hand, the miR-515 family members in general down regulated SNAI2expression, which was associated with up regulation of CDH1 expression.These remarkable differences may underlie two different mechanisms ofEMT regulation by the miR-200 and miR-515 family members. Expression andfunction of the 3 miR-515 family members has not been studied andreported in the publically available databases, and therefore provides afirst insight over the role of these miR in EMT.

Example 2 Materials and Methods Cell Culture

TSUpr1/pEcad-luc/Rluc (a.k.a. TSUpr1-pEcad) cells and the PC-3 prostatecancer cell line (ATCC# CRL-1435) were maintained in RPMI-1640 medium(Invitrogen, 31870), supplemented with 10% Fetal Bovine Serum (Sigma,F7524), L-Glutamine (Invitrogen 25030-024) and for TSUpr1/pEcad-luc/Rluc50 μg/ml Zeocin (Invitrogen, R250-01). Cells were maintained in ahumidified atmosphere at 37° C. and 5% CO₂.

Generation of an Inducible miRNA Expression System

To facilitate long-term and controlled miRNA expression, use was made ofthe Tet-inducible miR-X miRNA expression system (Clontech Inc.). ThemiRNA precursor was cloned in the 3′UTR of the ZsGreen fluorescentprotein transcription unit. MiRNA expression is governed in the vectorby the tightly regulated, inducible promoter Ptight, and the activity ofthe co-expressed transactivator (Tet-on). In the presence of doxycyclinethe Ptight promoter will be activated, resulting in the co-expression ofhigh levels of miRNA and ZsGreen1 protein, with low levels of expressionin non-induced cells.

The miR-200c and miR-520f precursors were PCR amplified, using proviralDNA of lentivirus-infected TSUpr1 cells as a template, and the universalpCDH forward and reverse vector-specific primers (SEQ ID NO: 84-85). Theamplification products were digested with EcoRI/NheI; these sites arefrom the lentiviral pCDH vector multiple cloning site. The digestedfragments were cloned into the EcoRI/NheI sites of a modified pEGFP-N3vector (Clontech #6080-1), which was obtained by BamHI/NotI excizing theEGFP ORF from the vector, and closing the vector after

Klenow DNA polymerase-mediated filling-in of the BamHI and Nonoverhangs. The miR precursor was excised from this vector byEagI/NheI-digestion and cloned into the EagI/NheI sites of thepmRi-ZsGreen1 vector (Clontech PT5049-1; FIG. 5A). Proper cloning wasconfirmed by DNA sequence analysis. The pTet-on transactivator vector(Clontech PT3899-5) was transfected into the PC3 prostate cancer cellline (ATCC# CRL-1435). G418-resistant clones were tested for properpTet-on activity, in a transient pTRE-luciferase-reporter assay.

Next, the pmRi-ZsGreen1-miR-X vectors were transfected into thePC3-Tet-on cells (clone 8), along with a puromycin selection marker.Puromycin-resistant colonies were selected, and quick-screened forDOX-inducible ZsGreen1 expression (by fluorescence measurement in 96well plates on the Victor3 multimeter). ZsGreen1-positive cells werefurther analyzed for DOX-inducible miRNA expression.

Cell Invasion Assays

For cell invasion assays, DOX-inducible PC3-mRi-ZsGreen1-miR-X cellswere incubated in the presence of DOX (1 ug/ml) for 2 days, prior to theinvasion assay. PC3 and TSUpr1-pEcad cells were transduced withmiR-expressing lentiviral particles (MOI=30), as described in example 1.The lentiviral transduced cells were puromycin-selected and passaged 1time before use. Fifty thousand (PC3) or 40,000 (TSUpr1-pEcad) cellswere seeded into Biocoat Matrigel Invasion chambers (8 micron; BD354480) in serum-free medium (FIG. 6A). The invasion chamber was placedin a 24-well containing medium with 10% fetal calf serum aschemo-attractant. As a control, the same amount of cells was seeded in24-well culture plates. After 48 hours incubation, cells in the invasionchamber were removed by aspiration and cleaning the inner compartmentwith a cotton swab. The invasion chamber was then put into CellTiter-GLO(CTG, Promega-G7571) cell viability reagent, incubated for 15 minutes,and then analyzed on a Victor3 luminometer. Cell invasion was calculatedas the CTG activity on the lower part of the membrane divided by the CTGactivity of the cells grown in a 24 well plate. Inhibition of cellinvasion by a specific miRNA was calculated by dividing the percentageof cell invasion of DOX-treated cells versus untreated cells, orinvasion of transduced cells versus empty-vector-transduced cells.

Real Time RT-PCR

The same protocol as described under example 1 was used to RT-PCR thefollowing genes: vimentin and CDH11 and HPRT. The following primers wereused for the respective genes:

HPRT forward 5′-CTCAACTTTAACTGGAAAGAATGTC-3′ reverse5′-TCCTTTTCACCAGCAAGCT-3′ Vimentin forward 5′-GGCTCAGATTCAGGAACAGC-3′reverse 5′-GCTTCAACGGCAAAGTTCTC-3′ CDH11 forward5′-GGTCTGGAACCAGTTCTTCG-3′ reverse 5′-GGCATGAATGTTCCCTGATT-3′(SEQ ID NO: 112-117)

Results

Validation of the MET-Inducing microRNAsIn Vitro Validation Using microRNA Mimics

To validate the microRNAs identified by screening a lentiviral miRNAexpression library, synthetic miRNA mimics were used. Synthetic miRmimics used were supplied by Ambion or Dharmacon (Thermo Scientific);see Table 7 for details. MiR-200c mimics of both companies were comparedand showed equal efficacy (i.e. CDH1 induction; data not shown).TSUpr1-pEcad-luc/Rluc cells were transiently transfected with 20 to 60nM of synthetic miR mimics. Four days post-transfection, Luc and RLucactivity were measured, and the Luc/RLuc ratios were normalized fornegative control (NC) mimic-transfected cells. Of all of the mimicstested, miR-124, 181a, 200c, 206 and miR-520f showed an induction of theLuc/RLuc ratio (FIG. 4A). Except for miR-200c and miR-520f, the othermiR mimics downregulated RLuc activity, and displayed cellular toxicity(not shown). Of all these miRs, miR-200c and miR-520f strongly induced(˜20-fold) endogenous CDH1 expression, whilst miR-124* and miR-181ainduced CDH1 about 2.5-fold (FIG. 4B). MiR-200c, miR-518 and miR-524downregulated CDH2 expression more than 30% (FIG. 4B). The efficacy ofthe miR mimics was further tested in several other tumor cell lines witha mesenchymal-like phenotype (based on the CDH1 and vimentin mRNAexpression ratios; FIG. 3). Like in TSUpr1-pEcad-luc/Rluc, miR-200c andmiR-520f also induced CDH1 expression in wild-type TSUpr1, PC3, PANC-1,MIA-PaCa2, and MDA-MB-231 cells (FIG. 4C).

Generation of an Inducible miRNA Expression System

G418-resistant clones were tested for proper pTet-on activity, in atransient pTRE-luciferase-reporter assay. Upon doxycycline (DOX)treatment (0.1 and 1.0 ug/ml), clone 8 showed a strong induction ofluciferase expression, whilst Luc activity in non-induced cells (no DOX)did not exceed background levels (FIG. 5B). As shown in FIG. 5C, severalpmRi-ZsGreen1-miR-X transfected clones displayed DOX-inducible miR-200cor miR-520f expression, which was DOX dose dependent, reaching maximumlevels at about 1 ug/ml DOX (not shown).

Endogenous gene expression in the inducible miR-200c and miR-520fexpressing cells was analyzed by qPCR. Upregulation of CDH1 wasobserved, whilst downregulation of CDH11 and vimentin was observedrepeatedly for both miRs (Table 8). PC3 cells also showed the weakestinduction of CDH1 by miR-200c and miR-520f mimics of all cell linestested. In addition to the down-regulation of mesenchymal markers,transcriptional repressors, such as ZEB1, ZEB2 and SNAI2 were alsodownregulated after induction of miR-200c and miR-520f (Table 8).Collectively, these data indicate that in the miR-200c and miR-520finducible cell lines, the corresponding miRs as well as their direct andindirect EMT target genes can be regulated at the molecular level.

Inhibition of Tumor Cell Invasion

To study whether the identified miRs also regulate EMT at the cellularlevel, cell invasion assays were performed using the miR-520f andmiR-200c inducible stable clones as generated above, and miR-200c andmiR-520f expressing lentivirus as described above. All miR-200c andmiR-520f clones tested, inhibited cell invasion in the range of 48-68%(FIG. 6B). Induction of miRNA expression was determined in parallelcultures and found positive (FIG. 6C). MiR levels did not show asignificant correlation with invasion percentage. These data indicatethat miR-520f, identified in the Luc screening assay as an MET-inducingmiRNA, could also revert one of the key cell biological aspects of EMT,namely cell invasion.

To confirm the effect of the other miR-515 family members, miR-518b andmiR-524, on cell invasion was tested in the TSUpr1/pEcad-luc/Rlucscreening model. TSUpr1-pEcad cells were infected with miR-precursorcontaining lentivirus. All three miRs belonging to the same miR-515family, miR-518b, miR-520f and miR-524, inhibited cell invasion by25-53% (FIG. 6D). Expression of the corresponding mature miRNAs wasconfirmed by stem-loop qPCR (as described in example 1; not shown).

Conclusions

The three miR-515 family members, miR-518b, miR-520f and miR-524, wereidentified in the MET screening. Mimics for miR-518b and miR-524downregulated the expression of the mesenchymal marker CDH2, whereas,miR-520f mimics strongly upregulated the expression of the epithelialmarker CDH1 in several cell line models. Cell invasion of PC3 cellsthrough Matrigel was inhibited after induction of miR-520f expression.The anti-invasion effect was also observed when all three miRNAs wereexpressed in the TSUpr1-pEcad screening model. In fact, miR-200c did notreduce invasion in this model. This indicates the great potentcy ofthese three miR-515 members as having anti-invasion activity in severalcell and tumor types in contrast to miR-200c.

Inhibition of Metastasis Formation in an Animal Model

The fact that these three miR-515 family members could activate theE-cadherin gene promoter in vitro, based on luciferase reporteractivation, and endogenous CDH1 induction (miR-520f) or decrease CDH2expression (miR-518b and miR-524), and could all inhibit tumor cellinvasion in vitro, indicates that these microRNAs are potent tools toinhibit the invasion and metastasis of tumor cells in vivo. To confirmthe anti-metastatic activity of miR-520f, miR-518b and miR-524 in an invivo setting, an experimental tumor metastasis experiment was set up.Male, immunodeficient, BALB/c nude mice (6-8 weeks old) are injectedwith 0.5×106 (200 μl volume) PC3-mRi-ZsGreen1cells expressing eithermiR-520f, miR-518b and miR-524, via the (lateral) tail vein (29). Groupsof 6 mice are given DOX-containing (0.2 mg/ml) drinking water during theentire experiment, and a control group of mice injected with non-DOXtreated PC3-mRi-ZsGreen1cells expressing either miR-520f, miR-518b andmiR-524 receive DOX-free water. Mice are monitored daily, and if notsuffering from serious inconvenience, are sacrificed after 2 months. Thelungs are the primary site of metastasis, since it contains the firstcapillary bed the injected cells will encounter after injection (30,31). It is suggested that cells passing the lung capillary bed, may alsoform metastasis in other organs (30). The number and size of metastasisare determined macroscopically (i.e. by visual examination through adissection microscope) and microscopically (i.e. by studying ZsGreenexpression in lung and other tissue sections). It is anticipated, basedon all available data presented above, that cells expressing eithermiR-520f, miR-518b or miR-524 form less (and smaller) metastasis in thisanimal model than cells that do not express said miRNAs. This proof ofconcept will pave the way for further pre-clinical testing of miR-520f,miR-518b and miR-524 as a drug to treat EMT-associated diseases.

To further confirm the inhibitory effect of miR-520f, miR-518b andmiR-524 on metastasis in vivo, different tumor cell lines, engineeredwith pmRi-ZsGreen1-miR-520f, 518b or 524, are orthotopically injectedinto nude mice (NOD-SCID or BALB/c nu/nu). At different times aftertumor cell injection, mice receive DOX-containing or DOX-free water.Local invasion and distant metastasis to the lungs and other organs aremonitored by in vivo imaging of ZsGreen1 or LUC. Over-expression ofmiR-520f, miR-518b or miR-524 (induced by DOX in the drinking water) isexpected to reduce the number of metastases. Moreover, miceorthotopically injected with tumor cells, in which miR-520f, miR-518b ormiR-524 was activated, are expected to have a mean survival rate thatwill be significantly prolonged compared with that of mice not receivingDOX (i.e. no miR-520f, miR-518b or miR-524 activation in tumor cells).Survival of the animals will be defined by serious complication-freesurvival.

TABLE 1 List of microRNAs identified by MET (Luciferase) screening andregulation of endogenous EMT-associated genes. TSUpr1- pEcad-Luc cellswere infected with 30 luciferase-positive microRNAs (see text). TotalRNA was reverse-transcribed, and used for SYBR Green real-time PCRanalysis of the CDH1 (Ecad), CDH2 (Ncad), SNAI1, SNAI2, ZEB1 and ZEB2genes. LUC, induction of FLuc/RLuc ratio, average of 3 experi- ments;CDH1, (induction of) endogenous CDH1 expression; CDH2, (inhibition of)endogenous CDH2 expression; Repressor, CDH1 transcriptional repressorsthat were down regulated at least 2-fold, or between 1.3 and 2-fold(underlined). Only data for 12 microRNAs that are of interest forfurther studies are shown; see text for a detailed reasoning ofselecting these 12 miRs. miRNA LUC CDH1 CDH2 CDH1/CDH2 RepressormiR-124-1 1.76 1.30 2.46 0.53 SNAI2 miR-181a-1 1.59 4.22 0.89 4.74 SNAI2miR-141 1.95 0.58 0.04 14.45 ZEB2, SNAI1 miR-200a 1.66 2.18 0.67 3.25ZEB2 miR-200c 2.53 0.71 0.04 17.65 ZEB2, ZEB1 miR-205 1.79 0.39 0.084.86 ZEB2 miR-429 1.50 3.39 0.41 8.28 ZEB2 miR-206 1.73 2.24 2.07 1.08SNAI2 miR-518b 1.62 3.09 1.67 1.85 SNAI2 miR-520f 1.43 12.92 1.16 11.14SNAI2, ZEB2 miR-524 1.57 1.60 0.45 3.56 ZEB2 miR-200b 2.08 5.17 1.184.38 ZEB2

TABLE 2 Precursor sequences of miRNAs identified inMET screening (see table 1) List of miRNA precursor sequences (5′ to 3′direction). SEQ ID miRNA Precursor sequence 22 miR-124-1AGGCCUCUCUCUCCGUGUUCACAGCGGACCUUGAUUUAAAUGUCCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAAUGGGGCUG 23 miR-124-2AUCAAGAUUAGAGGCUCUGCUCUCCGUGUUCACAGCGGACCUUGAUUUAAUGUCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGCGGAGC CUACGGCUGCACUUGAA 24miR-124-3 UGAGGGCCCCUCUGCGUGUUCACAGCGGACCUUGAUUUAAUGUCUAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGAGGCGCCUCC 25 miR-181a-1UGAGUUUUGAGGUUGCUUCAGUGAACAUUCAACGCUGUCGGUGAGUUUGGAAUUAAAAUCAAAACCAUCGACCGUUGAUUGUACCCUAUGGC UAACCAUCAUCUACUCCA 26miR-141 CGGCCGGCCCUGGGUCCAUCUUCCAGUACAGUGUUGGAUGGUCUAAUUGUGAAGCUCCUAACACUGUCUGGUAAAGAUGGCUCCCGGGUG GGUUC 27 miR-200aCCGGGCCCCUGUGAGCAUCUUACCGGACAGUGCUGGAUUUCCCAGCUUGACUCUAACACUGUCUGGUAACGAUGUUCAAAGGUGA CCCGC 28 miR-200cCCCUCGUCUUACCCAGCAGUGUUUGGGUGCGGUUGGGAGUCUC UAAUACUGCCGGGUAAUGAUGGAGG 29miR-205 AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGA AGUUCAGGAGGCAUGGAGCUGACA 30miR-429 GCGUCUUACCAGACAUGGUUAGACCUGGCCCUCUGUCUAAUACUGUCUGGUAAAACCGUCCAUCCGCUGC 31 miR-206UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAUUACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGUUUCGGCAAGUG 32 miR-518bUCAUGCUGUGGCCCUCCAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAACAAAGCGCUCCCCUUUAGAGGUUUACGGUUUGA 33 miR-520fUCUCAGGCUGUGACCCUCUAAAGGGAAGCGCUUUCUGUGGUCAGAAAGAAAAGCAAGUGCUUCCUUUUAGAGGGUUACCGUUU GGGA 34 miR-524UCUCAUGCUGUGACCCUACAAAGGGAAGCACUUUCUCUUGUCCAAAGGAAAAGAAGGCGCUUCCCUUUGGAGUGUUACGGUUUGAGA 35 miR-200bCCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGGAGUCAGGUCUCUAAUACUGCCUGGUAAUGAUGACGGCGGAGCCCUGC ACG All sequences wereobtained from miRBase (release 14:Sept 2009; www.mirbase.org).

TABLE 3 Mature sequences of miRNAs identified inMET screening (see table 1) List of mature miRNA sequences  (5′ to 3′direction). mature SEQ microRNA miRNA ID SEQ mature miRNA hsa-miR-miR-124 2 uaaggcacgcggugaaugcc 124-1 hsa-miR- miR-124* 3cguguucacagcggacc 124-2 uugau hsa-miR- 124-3 hsa-miR- miR-181a 4aacauucaacgcugucggugagu 181a-1 miR-181a* 5 accaucgaccguugauuguacchsa-miR- miR-141 6 uaacacugucugguaaagaugg 141 miR-141* 7caucuuccaguacaguguugga hsa-miR- miR-200a 8 uaacacugucugguaacgaugu 200amiR-200a* 9 caucuuaccggacagugcugga hsa-miR- miR-200b 10uaauacugccugguaaugauga 200b miR-200b* 11 caucuuacugggcagcauugga hsa-miR-miR-200c 12 uaauacugccggguaaugaugga 200c miR-200c* 13cgucuuacccagcaguguuugg hsa-miR- miR-205 14 uccuucauuccaccggagucug 205miR-205* 15 gauuucaguggagugaaguuc hsa-miR- miR-429 16uaauacugucugguaaaaccgu 429 hsa-miR- miR-206 17 uggaauguaaggaagugugugg206 hsa-miR- miR-518b 18 caaagcgcuccccuuuagaggu 518b hsa-miR- miR-520f19 aagugcuuccuuuuagaggguu 520f hsa-miR- miR-524-5p 20cuacaaagggaagcacuuucuc 524 miR-524-3p 21 gaaggcgcuucccuuuggagu Allsequences were obtained from miRBase (release 14: Sept 2009;www.mirbase.org).

TABLE 4 Sequences of miRNAs identified in MET screening as clonedin lentiviral vectors (see table 1) Seq ID miRNACloned sequence in lentiviral vector 36 miR-124-1TTTCTTTCACCTTTCCTTCCTTCCTTCCTCCTTTCCTTCCTCAGGAGAAAGGCCTCTCTCTCCGTGTTCACAGCGGACCTTGATTTAAATGTCCATACAATTAAGGCACGCGGTGAATGCCAAGAATGGGGCTGGCTGAGCACCGTGGGTCGGCGAGGGCCCGCCAAGGAAGGAGCGACC 37 miR-GTTGTTTCTGTCTCCCATCCCCTTCAGATACTTACAGATACTGTAAAG 181a-1TGAGTAGAATTCTGAGTTTTGAGGTTGCTTCAGTGAACATTCAACGCTGTCGGTGAGTTTGGAATTAAAATCAAAACCATCGACCGTTGATTGTACCCTATGGCTAACCATCATCTACTCCATGGTGCTCAGAATTCGCTGAAG ACAGGAAACCAAA 38miR-141 CCTGTAGCAACTGGTGAGCGCGCACCGTAGTTCTCTGTCGGCCGGCCCTGGGTCCATCTTCCAGTACAGTGTTGGATGGTCTAATTGTGAAGCTCCTAACACTGTCTGGTAAAGATGGCTCCCGGGTGGGTTCTCTCGGCAGTAACCTTCAGGGAGCCCTGAAGACCATGGAGGACTACTGACCAACAA CCTCTGACCTT 39 miR-200aGTTCTTCCCTGGGCTTCCACAGCAGCCCCTGCCTGCCTGGCGGGACCCCACGTCCCTCCCGGGCCCCTGTGAGCATCTTACCGGACAGTGCTGGATTTCCCAGCTTGACTCTAACACTGTCTGGTAACGATGTTCAAAGGTGACCCGCCGCTCGCCGGGGACACCACCGAGGCACATCCGGAGCTCCTAC T 40 miR-200cAAGCTGCCTGACCCAAGGTGGGCGGGCTGGGCGGGGGCCCTCGTCTTACCCAGCAGTGTTTGGGTGCGGTTGGGAGTCTCTAATACTGCCGGGTAATGATGGAGGCCCCTGTCCCTGTGTCAGCAACATCCATCGCCTCA 41 miR-205AGTGTCTACAGGCTGAGGTTGACATGCATCCCCACCCTCTGAGAAAAAGATCCTCAGACAATCCATGTGCTTCTCTTGTCCTTCATTCCACCGGAGTCTGTCTCATACCCAACCAGATTTCAGTGGAGTGAAGTTCAGGAGGCATGGAGCTGACAACCATGAGGCCTCGGCAGCCACCGCCACCACCGCCGCCGCCACCACCGTAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA GCAGCAAGAGTAACT 42miR-429 AGACACCAGCCCAGGACCCGGAGGCCACCCACACCACCGCCGGCCGATGGGCGTCTTACCAGACATGGTTAGACCTGGCCCTCTGTCTAATACTGTCTGGTAAAACCGTCCATCCGCTGCCTGATCACCGTTAGAGGAGAGAGCTGCCTGCCCTGCAGCTCATCAGTGCAAAGCC 43 miR-206GCAAGGAGGAAAGATGCTACAAGTGGCCCACTTCTGAGATGCGGGCTGCTTCTGGATGACACTGCTTCCCGAGGCCACATGCTTCTTTATATCCCCATATGGATTACTTTGCTATGGAATGTAAGGAAGTGTGTGGTTTCGGCAAGTGCCTCCTCGCTGGCCCCAGGGTACCACCCGGAGCACAGGTTTG GTGACCTTCTTC 44 miR-518bGCAAACAGGGCAAATAAATGCATCTTTATTTTGTGTCCATTTTAACCTGGTCAAGGAAAATTCCAACAGCAACATCAAAAAACCAGTGTTGGAGCAAGAATATGTCATGCTGTGGCCCTCCAGAGGGAAGCGCTTTCTGTTGTCTGAAAGAAAACAAAGCGCTCCCCTTTAGAGGTTTACGGTTTGAGTAAAGCAGCGTTGAAGTTGATGCTGATCTTGGTAATACATTTGCAGA GCGTGCTTATCATCAG 45miR-520f TGTGTCCATTTAAACCTGGTCAAGGAAGATTCCCACAAAAAATCCACGGTGCTGGAGCAAGAGGATCTCAGGCTGTGACCCTCTAAAGGGAAGCGCTTTCTGTGGTCAGAAAGAAAAGCAAGTGCTTCCTTTTAGAGGGTTACCGTTTGGGAAAAGCAATGTTGAAGTTGATGCTGATCTTGGTAAAATATTTGCAGAGCGTGCTTATCATCAG 46 miR-524CAAACAGGGCCAATAAATGCATCCTCATTTTTGTGTCCATTTTAACCTGGGCAAGGAAAATTCCAACAAAAAACCCAGAGTTCTGGAGCAAGAAGATCTCATGCTGTGACCCTACAAAGGGAAGCACTTTCTCTTGTCCAAAGGAAAAGAAGGCGCTTCCCTTTGGAGTGTTACGGTTTGAGAAAAGCAGCGTTGAAGTTGATGCTTATCTCGGTAATACATTTGTAGAGCATG CTTATCATGAGGCTTGGAC 47miR-200b CAGCCGGGCGGCCCCCGGACCCAGCTCGGGCAGCCGTGGCCATCTTACTGGGCAGCATTGGATGGAGTCAGGTCTCTAATACTGCCTGGTAATGATGACGGCGGAGCCCTGCACGCAGCGACCGGCCGACCCCGT

TABLE 5 Seed sequences of miRNAs identified inMET screening (see Table 1) List of miRNA seed sequences (5′ to 3′direction). mature SEQ Seed microRNA miRNA ID sequence hsa-miR-124-1miR-124 87 aaggcac hsa-miR-124-2 miR-124* 88 guguuca hsa-miR-124-3hsa-miR-181a-1 miR-181a 89 acauuca miR-181a* 90 ccaucga hsa-miR-141miR-141 91 aacacug miR-141* 92 aucuucc hsa-miR-200a miR-200a 93 aacacugmiR-200a* 94 aucuuac hsa-miR-200b miR-200b 95 aauacug miR-200b* 96aucuuac hsa-miR-200c miR-200c 97 aauacug miR-200c* 98 gucuuachsa-miR-205 miR-205 99 ccuucau miR-205* 100 auuucag hsa-miR-429 miR-429101 aauacug hsa-miR-206 miR-206 102 ggaaugu hsa-miR-518b miR-518b 103aaagcgc hsa-miR-520f miR-520f 104 agugcuu 105 aagugcu hsa-miR-524miR-524-5p 106 uacaaag miR-524-3p 107 aaggcgc All sequences wereobtained from miRBase (release 14: Sept 2009; www.mirbase.org). Seedsequence is defined as nucleotide 2-8 (5′ to 3′ direction) of thematuremiRNA sequence.

TABLE 6 IsomiR sequences of miRNAs identified in screening (see Table 1)Mature Seed miRNA (SEQ ID) (SEQ ID) IsomiR sequence hsa-miR-520fmir-520f AGUGCUU (104) AAGUGCUUCCUUUUAGAGGGU (118) AAGUGCU(105)AAGUGCUUCCUUUUAGAGGGUU^(*1) (119) CAAGUGCUUCCUUUUAGAGGGU (120)hsa-miR-518b mir-518b AAAGCGC (103) CAAAGCGCUCCCCUUUAGAGGU (121)CAAAGCGCUCCCCUUUAGAGG (122) CAAAGCGCUCCCCUUUAGAG (123) hsa-miR-524mir-524-3p AAGGCGC (107) GAAGGCGCUUCCCUUUGGAGUG (124)GAAGGCGCUUCCCUUUGGAGU (125) mir-524-5p UACAAAG (106)CUACAAAGGGAAGCACUUUCU (126) CUACAAAGGGAAGCACUUUCUC (127)CUACAAAGGGAAGCACUUUC (128) hsa-miR-124-1 mir-124 AAGGCAC (87)UAAGGCACGCGGUGAAUGCCAA (129) hsa-miR-124-2 UAAGGCACGCGGUGAAUGCCA (130)hsa-miR-124-3 UAAGGCACGCGGUGAAUGC (131) UAAGGCACGCGGUGAAUGCC^(*3) (132)hsa-miR-206 mir-206 GGAAUGU (102) UGGAAUGUAAGGAAGUGUGUGG (133)UGGAAUGUAAGGAAGUGUGUGGU (134) UGGAAUGUAAGGAAGUGUGUG (135) hsa-miR-mir-181a ACAUUCA (89) AACAUUCAACGCUGUCGGUGAGUUU (136) 181a-1AACAUUCAACGCUGUCGGUGAGU (137) AACAUUCAACGCUGUCGGUGAG (138)AACAUUCAACGCUGUCGGUGAGUU (139) AACAUUCAACGCUGUCGG (140)AACAUUCAACGCUGUCGGU (141) AACAUUCAACGCUGUCGGUG (142)AACAUUCAACGCUGUCGGUGA (143) hsa-miR-141 mir-141 AACACUG (91)UAACACUGUCUGGUAAAGAUGG (144) UAACACUGUCUGGUAAAGAUG (145)UAACACUGUCUGGUAAAGAUGGC (146) UAACACUGUCUGGUAAAGAU (147)UAACACUGUCUGGUAAAGA (148) UAACACUGUCUGGUAAAGAUGGCU (149) hsa-miR-200amir-200a AACACUG (93) UAACACUGUCUGGUAACGAUGUU (150UAACACUGUCUGGUAACGAUGU (151) hsa-miR-200b mir-200b AAUACUG (95)UAAUACUGCCUGGUAAUGAUGAC (152) UAAUACUGCCUGGUAAUGAUGA (153) mir-200b*AUCUUAC (96) CAUCUUACUGGGCAGCAUUGGA (154) CAUCUUACUGGGCAGCAUUGG (155)hsa-miR-200c mir-200c AAUACUG (97) UAAUACUGCCGGGUAAUGAUGGA (156)UAAUACUGCCGGGUAAUGAUGG (157) hsa-miR-205 mir-205 CCUUCAU (99)UCCUUCAUUCCACCGGAGUCU (158) UCCUUCAUUCCACCGGAGUCUG (159)UCCUUCAUUCCACCGGAGUCUGU (160) hsa-miR-429 mir-429 AAUACUG (101)UAAUACUGUCUGGUAAAACCGU (161) UAAUACUGUCUGGUAAAACCG (162)^(*1)miRBase-annotated sequence. Not detected in the deep-sequencinganalysis ^(*2)Only 0.9% of the total number of cloned sequences^(*3)Only 0.7% of the total number of cloned sequences These isomiRshave been detected after the analysis of 66 human tissue samples usinghigh-throughput deep sequencing and only isomiRs that represent >5% ofthe total number of cloned sequences are listed here, unless otherwiseindicated.

TABLE 7 List of microRNA mimics used for validation of MET screeningresults. Suppliers and product codes for all synthetic miR precurors areshown. All listed mimics are double stranded, with each strand having a100% complementary alignment with each other. One strand will be theexact sequence of the mature miRNA sequences as deposited in miRBase andlisted in table 3. The other strand contains certain modifications.miRNA mimic Supplier Product code Mimic Negative Control #1** Dharmacon*CN-001000-01-05 hsa-miR-200c miRIDIAN Mimic Dharmacon C-300646-05-0005hsa-miR-520f miRIDIAN Mimic Dharmacon C-300779-03-0005 hsa-miR-524-5pmiRIDIAN Mimic Dharmacon C-300806-03-0005 hsa-miR-518b miRIDIAN MimicDharmacon C-300798-03-0005 hsa-miR-124 Pre-miR precursor Ambion PM10691hsa-miR-124* Pre-miR precursor Ambion PM11154 hsa-miR-181a Pre-miRprecursor Ambion PM10421 hsa-miR-181a* Pre-miR precursor Ambion PM10381hsa-miR-200c Pre-miR precursor Ambion PM11714 hsa-miR-206 Pre-miRprecursor Ambion PM10409 *Dharmacon is now part of Thermo FisherScientific. **This negative control is based on C. Elegans Cel-miR-67(Seq ID 163).

TABLE 8 Gene expression in inducible microRNA transfected PC3 cells.PC3-Tet-on (clone 8) cells were stably transfected with pmRi-ZsGreen1-miR-X expression plasmids. Selected clones (see FIG. 5) weretreated with 1 μg/ml DOX for 4 days. Total RNA was isolated and geneexpression was studied by qPCR. Gene PC3-mRi-miR-200c PC3-mRi-miR-520fCDH1 = Up* CDH2 Weakly expressed Weakly expressed CDH11 Down* Down VIMDown n.d. ZEB1 Down Down ZEB2 Down = SNAI1 = = SNAI2 Down Down *morethan 1.5-fold; =, no change; n.d., not determined.

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ABBREVIATIONS

-   CDH1—E-cadherin-   CDH2—N-cadherin-   CDH11—OB-cadherin-   CTG—CEliTiter-GLO (cell viability assay)-   DOX—Doxycycline-   EMT—epithelial to mesenchymal transition-   miR—microRNA-   MET—mesenchymal to epithelial transition-   MOI—multiplicity of infection-   MTT—(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-   qPCR—quantitative PCR-   PCa—prostate cancer-   SNAI1—SNAIL homolog 1, alias SNAIL-   SNAI2—SNAIL homolog 2, alias SLUG-   TSU—TSUpr1-pEcad-Luc cells-   UBC—urinary bladder cancer-   VIM—vimentin-   ZEB1—zinc finger E-box binding homeobox 1, alias deltaEF1-   ZEB2—zinc finger E-box binding homeobox 2, alias SIP1

1. A method of treating a patient having a disease or a conditionassociated with Epithelial to Mesenchymal Transition (EMT), saidtreatment comprising administering a composition comprising an effectiveamount of a mature miRNA molecule comprising a minimum length of 6nucleotides and a maximum length of 50 nucleotides, comprising at least6 of the 7 nucleotides present in a seed sequence selected from thegroup consisting of SEQ ID NO: 103-107, and comprising at least 80%identity with a sequence selected from the group consisting of SEQ IDNO: 18-21 and 118-128; wherein said treatment results in amelioration ofa symptom of said disease or condition.
 2. The method of claim 1,wherein said treatment comprising further administering a compositioncomprising an effective amount of a mature miRNA molecule comprising aminimum length of 6 nucleotides and a maximum length of 50 nucleotides,comprising at least 6 of the 7 nucleotides present in a seed sequenceselected from the group consisting of SEQ ID NO: 87-102 and/orcomprising at least 80% identity with a sequence selected from the groupconsisting of SEQ ID NO: 2-17 and 129-162.
 3. The method of claim 1,wherein said miRNA molecule comprises at least 90% identity.
 4. Themethod of claim 1 wherein said miRNA molecule is a minimum of 10nucleotides in length.
 5. The method according to claim 1, wherein saidmiRNA molecule comprises a miRNA-518b, miRNA-520f and/or miRNA-524.
 6. Amethod of treating a patient having a disease or a condition associatedwith EMT, said treatment comprising administering a compositioncomprising an effective amount of a precursor of a miRNA molecule of50-400 nucleotides having at least 80% identity with a sequence selectedfrom the group consisting of SEQ ID NOs: 32-34 and 44-46; wherein saidtreatment results in amelioration of a symptom of said disease orcondition.
 7. The method of claim 6, wherein said treatment comprisingfurther administering a composition comprising an effective amount of amature miRNA molecule comprising a minimum length of 6 nucleotides and amaximum length a precursor of a miRNA molecule of 50-400 nucleotideshaving at least 80% identity with a sequence selected from the groupconsisting of SEQ ID NOs: 22-31, 35, 36-43 and
 47. 8. The method ofclaim 6, wherein said precursor RNA molecule is a minimum of 60nucleotides.
 9. The method of claim 1, wherein the disease or conditionassociated with EMT is a carcinoma and/or is not a leukemia and/or isnot a tissue sarcoma.
 10. The method of claim 9, wherein the carcinomais a brain, breast, colon, esophagus, gastrointestine, head, kidney,liver, lung, nasopharynx, neck, ovary, skin, stomach, testis, tongue, oruterus cancer.
 11. The method of claim 9, wherein said amelioration ofsaid carcinoma comprises: a decrease of proliferation of said tumorcells and/or an increase in said tumor cell death, an amelioration of asymptom of said carcinoma, wherein a tumor of said patient comprisescells which exhibit decreased expression of the E-cadherin (CDH1) generelative to a reference E-cadherin (CDH1) gene expression value.
 12. Themethod of claim 1, wherein said treatment further compriseschemotherapy, radiotherapy and/or surgery.
 13. The method of claim 1,wherein the disease or condition associated with EMT is poor woundhealing, diabetic renal nephropathy, allograft dysfunction, cataracts ordefects in cardiac valve formation.